Use of a pcv2 immunogenic composition for lessening clinical symptoms in pigs

ABSTRACT

The present invention relates to the use of an immunogenic composition that comprises a porcine circovirus type 2 (PCV2) antigen for treatment of several clinical manifestations (diseases). Preferably, the clinical manifestations are associated with a PCV2 infection. Preferably, they include lymphadenopathy, lymphoid depletion and/or multinucleated/giant histiocytes. Moreover, the clinical symptoms include lymphadenopathy in combination with one or a multiple of the following symptoms in pigs: (1) interstitial pneumonia with interlobular edema, (2) cutaneous pallor or icterus, (3) mottled atrophic livers, (4) gastric ulcers, (5) nephritis and (6) reproductive disorders, e.g. abortion, stillbirths, mummies, etc. Furthermore the clinical symptoms include Pia like lesions, normally known to be associated with Lawsonia intracellularis infections.

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 11/617,435,filed Dec. 28, 2006, which claims the benefit of provisional applicationSer. No. 60/755,016, filed Dec. 29, 2005 and provisional applicationSer. No. 60/829,809, filed Oct. 17, 2006, the teachings and contents allof which are hereby incorporated by reference.

SEQUENCE LISTING

This application contains a sequence listing in paper format and incomputer readable format, the teachings and content of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the use of an immunogenic compositioncomprising a porcine circovirus type 2 (PCV2) antigen for treatment ofseveral clinical manifestations (diseases). Preferably, those clinicalmanifestations are associated with a PCV2 infection. More particularly,the present invention is concerned with an immunological compositioneffective for providing an immune response that reduces, or lessens theseverity, of the clinical symptoms associated with PCV2 infection.Preferably, the immunological composition comprises a recombinantlyproduced antigen of PCV2. More preferably, the PCV2 antigen is arecombinantly produced protein encoded by one of the open reading frames(ORFs) in the PCV2 genome. Still more preferably, the antigen is PCV2ORF2 protein. Most particularly, the present invention is concerned withan immunological composition effective for treatment of clinicalsymptoms associated with PCV2 infections in swine receiving theimmunological composition, and wherein the composition comprises theprotein expressed by ORF2 of PCV2. Another aspect of the presentinvention is the use of any of the compositions provided herewith as amedicament, preferably as a veterinary medicament, even more preferablyas a vaccine. Moreover, the present invention also relates to the use ofany of the compositions described herein, for the preparation of amedicament for reducing or lessening the severity of clinical symptomsassociated with PCV2 infection. Preferably, the medicament is for theprevention of a PCV2 infection, even more preferably in swine. A furtheraspect of the present invention relates to a process for the productionof a medicament, comprising an immunogenic composition of PCV2 for thetreatment of several clinical manifestations.

2. Description of the Prior Art

Porcine circovirus type 2 (PCV2) is a small (17-22 nm in diameter),icosahedral, non-enveloped DNA virus, which contains a single-strandedcircular genome. PCV2 shares approximately 80% sequence identity withporcine circovirus type 1 (PCV1). However, in contrast with PCV1, whichis generally non-virulent, swine infected with PCV2 exhibit a syndromecommonly referred to as Post-weaning Multisystemic Wasting Syndrome(PMWS). PMWS is clinically characterized by wasting, paleness of theskin, unthriftiness, respiratory distress, diarrhea, icterus, andjaundice. In some affected swine, a combination of all symptoms will beapparent while other affected swine will only have one or two of thesesymptoms. During necropsy, microscopic and macroscopic lesions alsoappear on multiple tissues and organs, with lymphoid organs being themost common site for lesions. A strong correlation has been observedbetween the amount of PCV2 nucleic acid or antigen and the severity ofmicroscopic lymphoid lesions. Mortality rates for swine infected withPCV2 can approach 80%. In addition to PMWS, PCV2 has been associatedwith several other infections including pseudorabies, porcinereproductive and respiratory syndrome (PRRS), Glasser's disease,streptococcal meningitis, salmonellosis, postweaning colibacillosis,dietetic hepatosis, and suppurative bronchopneumonia. However, researchthus far has not confirmed whether any of these clinical symptoms are infact, the direct result of a PCV2 infection. Moreover, it is not yetknown whether any of these clinical symptoms can be effectively reducedor cured by an active agent directed against PCV2.

Current approaches to treat PCV2 infections include DNA-based vaccines,such as those described in U.S. Pat. No. 6,703,023. However, suchvaccines have been ineffective at conferring protective immunity againstPCV2 infection or reducing, lessening the severity of, or curing anyclinical symptoms associated therewith. Moreover, vaccines described inthe prior art were focused solely on the prevention of PCV2 infectionsin swine, but did not consider any further medical use.

Accordingly, what is needed in the art is an immunogenic composition forthe treatment of several clinical manifestations. Further, what isneeded in the art is an immunological composition which confersprotective immunity against PCV2 infection but which can also be used totreat existing clinical symptoms associated with PCV2 infection.

DISCLOSURE OF THE INVENTION

The present invention overcomes the problems inherent in the prior artand provides a distinct advance in the state of the art. The presentinvention provides a medicinal use(s) of immunogenic composition(s)comprising PCV2 antigen.

In general no adverse events or injection site reactions were noted forany of the PCV2 antigen immunogenic compositions as used herein. Thus,the immunogenic compositions used herein appear to be safe whenadministered to young pigs, preferably to pigs not older than 15 weeksof age, more preferably not older than 6 weeks of age, even morepreferably not older than 3 weeks, most preferably not older than 2weeks. Alternatively, it is preferred that the administration of theimmunogenic compositions of the present invention occur within at least2 and preferably within at least 3 weeks of exposure to virulent PCV.According to a further embodiment, the immunogenic compositions usedherein for any medicinal use described herein, is adminstered to pigs of3 weeks of age or older, preferably of 2 weeks of age or older, mostpreferably but not older than 15 weeks of age.

Unexpectedly, it was found that the therapeutic use of the immunogeniccompositions described below, is effective for lessening the severity ofvarious clinical symptoms in swine. In particular, it was discoveredthat the therapeutic use of the immunogenic compositions of the presentinvention, and specifically compositions comprising PCV2 ORF2 antigen,is effective for reducing or lessening lymphadenopathy, lymphoiddepletion and/or multinucleated/giant histiocytes in swine infected withPCV2. Moreover, the therapeutic use of an antigenic composition, asprovided herewith, and that comprises PCV2 antigen, preferably ORF2antigen, reduces the overall circovirus load and its immunosuppressiveimpact, thereby resulting in a higher level of general diseaseresistance and a reduced incidence of PCV-2 associated diseases andsymptoms.

Thus one aspect of the present invention relates to the use of animmunogenic composition comprising PCV2 antigen, preferably recombinantPCV2 antigen, and more preferably PCV2 ORF2 protein as providedherewith, for the preparation of a medicament for the prevention,lessening and/or reduction of lymphadenopathy, lymphoid depletion and/ormultinucleated/giant histiocytes in swine. Preferably, said medicamentis effective for the prevention, lessening and/or reduction oflymphadenopathy, lymphoid depletion and/or multinucleated/gianthistiocytes associated faith PCV2 infections in swine. Still morepreferably, said medicament is effective for the prevention, lesseningand/or reduction of lymphadenopathy, lymphoid depletion and/ormultinucleated/giant histiocytes associated with PCV2 infections inpigs, when administered to pigs not older than 1.5 weeks of age, morepreferably not older than 6 weeks of age, even more preferably not olderthan 3 weeks, and most preferably not older than 2 weeks. Alternatively,it is preferred that the administration of the immunogenic compositionsof the present invention occur within at least 2 and preferably withinat least 3 weeks of exposure to virulent PCV.

Another aspect of the present invention relates to a method for thetreatment of lymphadenopathy, lymphoid depletion and/ormultinucleated/giant histiocytes in swine, comprising the administrationof an immunogenic composition as provided herewith, to a pig, saidimmunogenic composition comprising a PCV2 antigen, preferably arecombinant PCV2 antigen, and more preferably PCV2 ORF2 protein. In yetanother aspect, the present invention provides a method for thetreatment of lymphadenopathy, lymphoid depletion and/ormultinucleated/giant histiocytes associated with a PCV2 infection inswine, comprising the administration of an immunogenic composition asprovided herewith, to a pig, said immunogenic composition comprising aPCV2 antigen, preferably a recombinant PCV2 antigen and more preferablyPCV2 ORF2 protein. Preferably, said treatment results in the lessening,reduction, prevention, and/or cure of the lymphadenopathy, lymphoiddepletion and/or multinucleated/giant histiocytes in swine receivingsaid immunogenic composition. According to a further aspect, saidmethods for treatment further comprise the administration of saidimmunogenic composition to pigs not older than 15 weeks of age, morepreferably not older than 6 weeks of age, even more preferably not olderthan 3 weeks, and most preferably not older than 2 weeks. Alternatively,it is preferred that the administration of the immunogenic compositionsof the present invention occur within at least 2 and preferably withinat least 3 weeks of exposure to virulent PCV.

It was further discovered that the therapeutic use of an immunogeniccomposition comprising PCV2 antigen, preferably a recombinant PCV2antigen, and most preferably PCV2 ORF2 protein, as provided herewith,can reduce or lessen lymphadenopathy in combination with one or amultiple of the following symptoms in affected swine: (1) interstitialpneumonia with interlobular edema, (2) cutaneous pallor or icterus, (3)mottled atrophic livers, (4) gastric ulcers, (5) nephritis and (6)reproductive disorders, e.g. abortion, stillbirths, mummies, etc.

Thus one aspect of the present invention relates to the use of animmunogenic composition comprising PCV2 antigen, preferably arecombinant PCV2 antigen and more preferably, PCV2 ORF2 protein asprovided herewith, for the preparation of a medicament for theprevention, lessening and/or reduction of lymphadenopathy in combinationwith one or a multiple of the following symptoms in pigs: (1)interstitial pneumonia with interlobular edema, (2) cutaneous pallor oricterus, (3) mottled atrophic livers, (4) gastric ulcers, (5) nephritisand (6) reproductive disorders, e.g. abortion, stillbirths, mummies,etc., in pigs. Preferably, said medicament is effective for theprevention, lessening and/or reduction of lymphadenopathy in combinationwith one or a multiple of the following symptoms associated with PCV2infection in pigs: (1) interstitial pneumonia with interlobular edema,(2) cutaneous pallor or icterus, (3) mottled atrophic livers, (4)gastric ulcers, (5) nephritis and (6) reproductive disorders, e.g.abortion, stillbirths, mummies, etc. According to a further aspect, saidmedicament is effective for the prevention, lessening and/or reductionof lymphadenopathy in combination with one or a multiple of thefollowing symptoms in pigs: (1) interstitial pneumonia with interlobularedema, (2) cutaneous pallor or icterus, (3) mottled atrophic livers, (4)gastric ulcers, (5) nephritis and (6) reproductive disorders, e.g.abortion, stillbirths, mummies, etc., in pigs, when administered to pigsnot older than 15 weeks of age, more preferably not older than 6 weeksof age even more preferably not older than 3 weeks, and most preferablynot older than 2 weeks. Alternatively, it is preferred that theadministration of the immunogenic compositions of the present inventionoccur within at least 2 and preferably within at least 3 weeks ofexposure to virulent PCV.

Moreover, the present invention also relates to a method for thetreatment of lymphadenopathy in combination with one or a multiple ofthe following symptoms in pigs: (1) interstitial pneumonia withinterlobular edema, (2) cutaneous pallor or icterus, (3) mottledatrophic livers, (4) gastric ulcers, (5) nephritis and (6) reproductivedisorders, e.g. abortion, stillbirths, mummies, etc., said methodcomprising the administration of an immunogenic composition comprisingPCV2 antigen, preferably a recombinant PCV2 antigen, and more preferablyPCV2 ORF2 protein as provided herewith. Preferably, the presentinvention also relates to a method for the treatment of lymphadenopathyin combination with one or a multiple of the following symptomsassociated with PCV2 infection in pigs: (1) interstitial pneumonia withinterlobular edema, (2) cutaneous pallor or icterus, (3) mottledatrophic livers, (4) gastric ulcers, (5) nephritis and (6) reproductivedisorders, e.g. abortion, stillbirths, mummies, etc., said methodcomprising the administration of an immunogenic composition comprisingPCV2 antigen, preferably recombinant PCV2 antigen and more preferablyPCV2 ORF2 protein, as provided herewith, to a pig. Preferably, saidtreatment results in the lessening or reduction of the lymphadenopathy,and one or multiple of the following symptoms associated with PCV2infection in pigs: (1) interstitial pneumonia with interlobular edema,(2) cutaneous pallor or icterus, (3) mottled atrophic livers, (4)gastric ulcers, (5) nephritis and (6) reproductive disorders, e.g.abortion, stillbirths, mummies, etc. According to a further aspect, saidmethods for treatment further comprise administration of the immunogeniccomposition comprising PCV2 antigen, preferably recombinant PCV2 antigenand more preferably PCV2 ORF2 protein, as provided herein, to pigs notolder than 15 weeks of age, more preferably not older than 6 weeks ofage, even more preferably not older than 3 weeks, and most preferablynot older than 2 weeks. Alternatively, it is preferred that theadministration of the immunogenic compositions of the present inventionoccur within at least 2 and preferably within at least 3 weeks ofexposure to virulent PCV.

It was also unexpectedly found that the therapeutic use of animmunogenic composition comprising PCV antigen, preferably recombinantPCV2 antigen and more preferably PCV2 ORF2 protein as provided herewith,can also reduce or lessen Pia like lesions, normally known to beassociated with Lawsonia intracellularis infections (Ileitis).

Thus one aspect of the present invention relates to the use of animmunogenic composition comprising PCV2 antigen, preferably recombinantPCV2 antigen and more preferably PCV2 ORF2 protein as provided herewith,for the preparation of a medicament for the prevention, lessening theseverity of and/or reduction of Pia like lesions, normally known to beassociated with Lawsonia intracellularis infections in swine. Accordingto a further aspect, said medicament is effective for the prevention,lessening of the severity of and/or reduction of Pia like lesions,normally known to be associated with Lawsonia intracellularisinfections, when administered to pigs not older than 15 weeks of age,more preferably not older than 6 weeks of age, even more preferably notolder than 3 weeks, and most preferably not older than 2 weeks.Alternatively, it is preferred that the administration of theimmunogenic compositions of the present invention occur within at least2 and preferably within at least 3 weeks of exposure to virulent PCV.

Moreover, the present invention also relates to a method for thetreatment of Pia like lesions, normally known to be associated withLawsonia intracellularis infections, said method comprising theadministration of an immunogenic composition comprising PCV2 antigen,preferably recombinant PCV2 antigen and more preferably PCV2 ORF2protein as provided herein, to a pig. Preferably, said treatment resultsin the lessening or reduction of the Pia like lesions, normally known tobe associated with Lawsonia intracellularis infections. According to afurther aspect, the methods for treatment described above furthercomprise the administration of the immunogenic composition comprisingPCV2 antigen, preferably recombinant PCV2 antigen, and more preferablyPCV2 ORF2 protein as provided herein, to pigs not older than 15 weeks ofage, more preferably not older than 6 weeks of age, even more preferablynot older than 3 weeks, and most preferably not older than 2 weeks.Alternatively, it is preferred that the administration of theimmunogenic compositions of the present invention occur within at least2 and preferably within at least 3 weeks of exposure to virulent PCV.

The Immunogenic Composition

The immunogenic composition as used herein is effective for inducing animmune response against PCV2 and preventing, reducing and/or lesseningthe severity of the clinical symptoms associated with PCV2 infection.The composition generally comprises at least one PCV2 antigen.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. The term “immunogeniccomposition” as used herein refers to any pharmaceutical compositioncontaining a PCV2 antigen, which composition can be used to prevent ortreat a PCV2 infection-associated disease or condition in a subject. Apreferred immunogenic composition can induce, stimulate or enhance theimmune response against PCV2. The term thus encompasses both subunitimmunogenic compositions, as described below, as well as compositionscontaining whole killed, or attenuated and/or inactivated PCV2.

The term “subunit immunogenic composition” as used herein refers to acomposition containing at least one immunogenic polypeptide or antigen,but not all antigens, derived from or homologous to an antigen fromPCV2. Such a composition is substantially free of intact PCV2. Thus, a“subunit immunogenic composition” is prepared from at least partiallypurified or fractionated (preferably substantially purified) immunogenicpolypeptides from PCV2, or recombinant analogs thereof. A subunitimmunogenic composition can comprise the subunit antigen or antigens ofinterest substantially free of other antigens or polypeptides from PCV2,or in fractionated from. A preferred immunogenic subunit compositioncomprises the PCV2 ORF2 protein as described below.

An “immunological or immune response” to a composition or vaccine is thedevelopment in the host of a cellular and/or antibody-mediated immuneresponse to the composition or vaccine of interest. Usually, an “immuneresponse” includes but is not limited to one or more of the followingeffects: the production or activation of antibodies, B cells, helper Tcells, suppressor T cells, and/or cytotoxic T cells and/or yd T cells,directed specifically to an antigen or antigens included in thecomposition or vaccine of interest. Preferably, the host will displayeither a therapeutic or protective immunological response such thatresistance to new infection will be enhanced and/or the clinicalseverity of the disease reduced. Such protection will be demonstrated byeither a reduction in number or severity of, or lack of one or more ofthe symptoms associated with PCV2 infections as described above.

The terms “immunogenic” protein or polypeptide or “antigen” as usedherein refer to an amino acid sequence which elicits an immunologicalresponse as described above. An “immunogenic” protein or polypeptide, asused herein, includes the full-length sequence of any PCV2 proteins,analogs thereof, or immunogenic fragments thereof. The term “immunogenicfragment” refers to a fragment of a protein which includes one or moreepitopes and thus elicits the immunological response described above.Such fragments can be identified using any number of epitope mappingtechniques, well known in the art. See, e.g., Epitope Mapping Protocolsin Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996)Humana Press, Totowa, N.J. For example, linear epitopes may bedetermined by e.g., concurrently synthesizing large numbers of peptideson solid supports, the peptides corresponding to portions of the proteinmolecule, and reacting the peptides with antibodies while the peptidesare still attached to the supports. Such techniques are known in the artand described in, e.g., U.S. Pat. No. 4,708,871; Geysen et al. (1984)Proc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al. (1986) Molec.Immunol. 23:709-715. Similarly, conformational epitopes are readilyidentified by determining spatial conformation of amino acids such asby, e.g., x-ray crystallography and 2-dimensional nuclear magneticresonance. See, e.g., Epitope Mapping Protocols, supra.

Synthetic antigens are also included within the definition, for example,polyepitopes, flanking epitopes, and other recombinant or syntheticallyderived antigens. See, e.g., Bergmann et al. (1993) Eur. J. Immunol.23:2777-2781; Bergmann et al. (1996), J. Immunol. 157:3242-3249;Suhrbier, A. (1997), Immunol. and Cell Biol. 75:402-408; Gardner et al.,(1998) 12th World AIDS Conference, Geneva, Switzerland, Jun. 28-Jul. 3,1998.

In a preferred embodiment of the present invention, an immunogeniccomposition that induces an immune response and, more preferably,confers protective immunity against the clinical signs of PCV2infection, is provided. The composition most preferably comprises thepolypeptide, or a fragment thereof, expressed by ORF2 of PCV2, as theantigenic component of the composition. PCV2 ORF2 DNA and protein, usedherein for the preparation of the compositions and within the processesprovided herein is a highly conserved domain within PCV2 isolates andthereby, any PCV2 ORF2 would be effective as the source of the PCV ORF2DNA and/or polypeptide as used herein. A preferred PCV2 ORF2 protein isthat of SEQ ID NO. 11. A preferred PCV ORF2 polypeptide is providedherein as SEQ ID NO. 5, but it is understood by those of skill in theart that this sequence could vary by as much as 6-10% in sequencehomology and still retain the antigenic characteristics that render ituseful in immunogenic compositions. The antigenic characteristics of animmunological composition can be, for example, estimated by thechallenge experiment as provided by Example 4. Moreover, the antigeniccharacteristic of a modified antigen is still retained, when themodified antigen confers at least 70%, preferably 80%, more preferably90% of the protective immunity as compared to the PCV2 ORF 2 protein,encoded by the polynucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4. An“immunogenic composition” as used herein, means a PCV2 ORF2 proteinwhich elicits an “immunological response” in the host of a cellularand/or antibody-mediated immune response to PCV2 ORF2 protein.Preferably, this immunogenic composition is capable of eliciting orenhancing an immune response against PCV2 thereby conferring protectiveimmunity against PCV2 infection and a reduction in the incidence of,severity of, or prevention of one or more, and preferably all of theclinical signs associated therewith.

In some forms, immunogenic portions of PCV2 ORF2 protein are used as theantigenic component in the composition. The term “immunogenic portion”as used herein refers to truncated and/or substituted forms, orfragments of PCV2 ORF2 protein and/or polynucleotide, respectively.Preferably, such truncated and/or substituted forms, or fragments willcomprise at least 6 contiguous amino acids from the full-length ORF2polypeptide. More preferably, the truncated or substituted forms, orfragments will have at least 10, more preferably at least 15, and stillmore preferably at least 19 contiguous amino acids from the full-lengthORF2 polypeptide. Two preferred sequences in this respect are providedherein as SEQ ID NOs. 9 and 10. It is further understood that suchsequences may be a part of larger fragments or truncated forms.

A further preferred PCV2 ORF2 polypeptide provided herein is encoded bythe nucleotide sequences of SEQ ID NO: 3 or SEQ ID NO: 4. However, it isunderstood by those of skill in the art that this sequence could vary byas much as 6-20% in sequence homology and still retain the antigeniccharacteristics that render it useful in immunogenic compositions. Insome forms, a truncated or substituted form, or fragment of this PVC2ORF2 polypeptide is used as the antigenic component in the composition.Preferably, such truncated or substituted forms, or fragments willcomprise at least 18 contiguous nucleotides from the full-length ORF2nucleotide sequence, e.g. of SEQ ID NO: 3 or SEQ ID NO: 4. Morepreferably, the truncated or substituted forms, or fragments, will haveat least 30, more preferably at least 45, and still more preferably atleast 57 contiguous nucleotides of the full-length ORF2 nucleotidesequence, e.g. SEQ ID NO: 3 or SEQ ID NO: 4.

“Sequence Identity” as it is known in the art refers to a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, namely a reference sequence and a given sequence to becompared with the reference sequence. Sequence identity is determined bycomparing the given sequence to the reference sequence after thesequences have been optimally aligned to produce the highest degree ofsequence similarity, as determined by the match between strings of suchsequences. Upon such alignment, sequence identity is ascertained on aposition-by-position basis, e.g., the sequences are “identical” at aparticular position if at that position, the nucleotides or amino acidresidues are identical. The total number of such position identities isthen divided by the total number of nucleotides or residues in thereference sequence to give % sequence identity. Sequence identity can bereadily calculated by known methods, including but not limited to, thosedescribed in Computational Molecular Biology, Lesk, A. N., ed., OxfordUniversity Press, New York (1988), Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York (1993); ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey (1994); Sequence Analysis in MolecularBiology, von Heinge, G., Academic Press (1987); Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York(1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988), the teachings of which are incorporated herein by reference.Preferred methods to determine the sequence identity are designed togive the largest match between the sequences tested. Methods todetermine sequence identity are codified in publicly available computerprograms which determine sequence identity between given sequences.Examples of such programs include, but are not limited to, the GCGprogram package (Devereux, J., et al., Nucleic Acids Research, 12(1):387(1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec.Biol., 215:403-410 (1990). The BLASTX program is publicly available fromNCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIHBethesda, Md. 20894, Altschul, S. F. et al., J. Molec. Biol.,215:403-410 (1990), the teachings of which are incorporated herein byreference). These programs optimally align sequences using default gapweights in order to produce the highest level of sequence identitybetween the given and reference sequences. As an illustration, by apolynucleotide having a nucleotide sequence having at least, forexample, 85%, preferably 90%, even more preferably 95% “sequenceidentity” to a reference nucleotide sequence, it is intended that thenucleotide sequence of the given polynucleotide is identical to thereference sequence except that the given polynucleotide sequence mayinclude up to 15, preferably up to 10, even more preferably up to 5point mutations per each 100 nucleotides of the reference nucleotidesequence. In other words, in a polynucleotide having a nucleotidesequence having at least 85%, preferably 90%, even more preferably 95%identity relative to the reference nucleotide sequence, up to 15%,preferably 10%, even more preferably 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 15%, preferably 10%, evenmore preferably 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. Analogously, by a polypeptide having a given aminoacid sequence having at least, for example, 85%, preferably 90%, evenmore preferably 95% sequence identity to a reference amino acidsequence, it is intended that the given amino acid sequence of thepolypeptide is identical to the reference sequence except that the givenpolypeptide sequence may include up to 15, preferably up to 10, evenmore preferably up to 5 amino acid alterations per each 100 amino acidsof the reference amino acid sequence. In other words, to obtain a givenpolypeptide sequence having at least 85%, preferably 90%, even morepreferably 95% sequence identity with a reference amino acid sequence,up to 15%, preferably up to 10%, even more preferably up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to15%, preferably up to 10%, even more preferably up to 5% of the totalnumber of amino acid residues in the reference sequence may be insertedinto the reference sequence. These alterations of the reference sequencemay occur at the amino or the carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in the one or more contiguous groups within thereference sequence. Preferably, residue positions which are notidentical differ by conservative amino acid substitutions. However,conservative substitutions are not included as a match when determiningsequence identity.

“Sequence homology”, as used herein, refers to a method of determiningthe relatedness of two sequences. To determine sequence homology, two ormore sequences are optimally aligned, and gaps are introduced ifnecessary. However, in contrast to “sequence identity”, conservativeamino acid substitutions are counted as a match when determiningsequence homology. In other words, to obtain a polypeptide orpolynucleotide having 95% sequence homology with a reference sequence,85%, preferably 90%, even more preferably 95% of the amino acid residuesor nucleotides in the reference sequence must match or comprise aconservative substitution with another amino acid or nucleotide, or anumber of amino acids or nucleotides up to 15%, preferably up to 10%,even more preferably up to 5% of the total amino acid residues ornucleotides, not including conservative substitutions, in the referencesequence may be inserted into the reference sequence. Preferably thehomolog sequence comprises at least a stretch of 50, even morepreferably at least 100, even more preferably at least 250, and evenmore preferably at least 500 nucleotides.

A “conservative substitution” refers to the substitution of an aminoacid residue or nucleotide with another amino acid residue or nucleotidehaving similar characteristics or properties including size,hydrophobicity, etc., such that the overall functionality does notchange significantly.

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide orpolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein.

Thus, the immunogenic composition as used herein also refers to acomposition that comprises PCV2 ORF2 protein, wherein said PCV2 ORF2protein is anyone of those, described above. Preferably, said PCV2 ORF2protein is

-   -   i) a polypeptide comprising the sequence of SEQ ID NO: 5, SEQ ID        NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11;    -   ii) any polypeptide that is at least 80% homologous to the        polypeptide of i),    -   iii) any immunogenic portion of the polypeptides of i) and/or        ii)    -   iv) the immunogenic portion of iii), comprising at least 10        contiguous amino acids included in the sequences of SEQ ID NO:        5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11,    -   v) a polypeptide that is encoded by a DNA comprising the        sequence of SEQ ID NO: 3 or SEQ ID NO: 4.    -   vi) any polypeptide that is encoded by a polynucleotide that is        at least 80% homologous to the polynucleotide of v),    -   vii) any immunogenic portion of the polypeptides encoded by the        polynucleotide of v) and/or vi)    -   viii) the immunogenic portion of vii), wherein polynucleotide        coding for said immunogenic portion comprises at least 30        contiguous nucleotides included in the sequences of SEQ ID NO:        3, or SEQ ID NO: 4.

Preferably any of those immunogenic portions have the immunogeniccharacteristics of PCV2 ORF2 protein that is encoded by the sequence ofSEQ ID NO: 3 or SEQ ID NO: 4.

According to a further aspect, PCV2 ORF2 protein is provided in theimmunological composition at an antigen inclusion level effective forinducing the desired immune response, namely reducing the incidence of,lessening the severity of, or preventing one or more clinical signsresulting from PCV2 infection. Preferably, the PCV2 ORF2 proteininclusion level is at least 0.2 μg antigen/ml of the final immunogeniccomposition (μg/ml), more preferably from about 0.2 to about 400 μg/ml,still more preferably from about 0.3 to about 200 μg/ml, even morepreferably from about 0.35 to about 100 μg/ml, still more preferablyfrom about 0.4 to about 50 μg/ml, still more preferably from about 0.45to about 30 μg/ml, still more preferably from about 0.6 to about 15μg/ml, even more preferably from about 0.75 to about 8 μg/ml, even morepreferably from about 1.0 to about 6 μg/ml, still more preferably fromabout 1.3 to about 3.0 μg/ml, even more preferably from about 1.4 toabout 2.5 μg/ml, even more preferably from about 1.5 to about 2.0 μg/ml,and most preferably about 1.6 μg/ml.

According to a further aspect, the ORF2 antigen inclusion level is atleast 0.2 μg/PCV2 ORF2 protein as described above per dose of the finalantigenic composition (μg/dose), more preferably from about 0.2 to about400 μg/dose, still more preferably from about 0.3 to about 200 μg/dose,even more preferably from about 0.35 to about 100 μg/dose, still morepreferably from about 0.4 to about 50 μg/dose, still more preferablyfrom about 0.45 to about 30 μg/dose, still more preferably from about0.6 to about 15 μg/dose, even more preferably from about 0.75 to about 8μg/dose, even more preferably from about 1.0 to about 6 μg/dose, stillmore preferably from about 1.3 to about 3.0 μg/dose, even morepreferably from about 1.4 to about 2.5 μg/dose, even more preferablyfrom about 1.5 to about 2.0 μg/dose, and most preferably about 1.6μg/dose.

The PCV2 ORF2 polypeptide used in the immunogenic composition inaccordance with the present invention can be derived in any fashionincluding isolation and purification of PCV2 ORF2, standard proteinsynthesis, and recombinant methodology. Preferred methods for obtainingPCV2 ORF2 polypeptide are provided in U.S. patent application Ser. No.11/034,797, the teachings and content of which are hereby incorporatedby reference. Briefly, susceptible cells are infected with a recombinantviral vector containing PCV2 ORF2 DNA coding sequences, PCV2 ORF2polypeptide is expressed by the recombinant virus, and the expressedPCV2 ORF2 polypeptide is recovered from the supernate by filtration andinactivated by any conventional method, preferably using binaryethylenimine, which is then neutralized to stop the inactivationprocess.

The immunogenic composition as used herein also refers to a compositionthat comprises i) any of the PCV2 ORF2 protein described above,preferably in concentrations described above, and ii) at least a portionof the viral vector expressing said PCV2 ORF2 protein, preferably of arecombinant baculovirus. Moreover, the immunogenic composition cancomprise i) any of the PCV2 ORF2 proteins described above, preferably inconcentrations described above, ii) at least a portion of the viralvector expressing said PCV2 ORF2 protein, preferably of a recombinantbaculovirus, and iii) a portion of the cell culture supernate.

The immunogenic composition as used herein also refers to a compositionthat comprises i) any of the PCV2 ORF2 proteins described above,preferably in concentrations described above, ii) at least a portion ofthe viral vector expressing said PCV2 ORF2 protein, preferably of arecombinant baculovirus, and iii) a portion of the cell culture; whereinabout 90% of the components have a size smaller than 1 μm.

The immunogenic composition as used herein also refers to a compositionthat comprises i) any of the PCV2 ORF2 proteins described above,preferably in concentrations described above, ii) at least a portion ofthe viral vector expressing said PCV2 ORF2 protein, iii) a portion ofthe cell culture, iv) and inactivating agent to inactivate therecombinant viral vector preferably BEI, wherein about 90% of thecomponents i) to iii) have a size smaller than 1 μm. Preferably, BEI ispresent in concentrations effective to inactivate the baculovirus.Effective concentrations are described above.

The immunogenic composition as used herein also refers to a compositionthat comprises i) any of the PCV2 ORF2 proteins described above,preferably in concentrations described above, ii) at least a portion ofthe viral vector expressing said PCV2 ORF2 protein, iii) a portion ofthe cell culture, iv) an inactivating agent to inactivate therecombinant viral vector preferably BEI, and v) an neutralization agentto stop the inactivation mediated by the inactivating agent, whereinabout 90% of the components i) to iii) have a size smaller than 1 μm.Preferably, if the inactivating agent is BEI, said composition comprisessodium thiosulfate in equivalent amounts to BEI.

The polypeptide is incorporated into a composition that can beadministered to an animal susceptible to PCV2 infection. In preferredforms, the composition may also include additional components known tothose of skill in the art (see also Remington's Pharmaceutical Sciences.(1990). 18th ed. Mack Publ., Easton). Additionally, the composition mayinclude one or more veterinary-acceptable carriers. As used herein, “aveterinary-acceptable carrier” includes any and all solvents, dispersionmedia, coatings, adjuvants, stabilizing agents, diluents, preservatives,antibacterial and antifungal agents, isotonic agents, adsorptiondelaying agents, and the like. In a preferred embodiment, theimmunogenic composition comprises PCV2 ORF2 protein as providedherewith, preferably in concentrations described above, which is mixedwith an adjuvant, preferably Carbopol, and physiological saline.

Those of skill in the art will understand that the composition usedherein may incorporate known injectable, physiologically acceptablesterile solutions. For preparing a ready-to-use solution for parenteralinjection or infusion, aqueous isotonic solutions, such as e.g. salineor corresponding plasma protein solutions, are readily available. Inaddition, the immunogenic and vaccine compositions of the presentinvention can include diluents, isotonic agents, stabilizers, oradjuvants. Diluents can include water, saline, dextrose, ethanol,glycerol, and the like. Isotonic agents can include sodium chloride,dextrose, mannitol, sorbitol, and lactose, among others. Stabilizersinclude albumin and alkali salts of ethylendiamintetracetic acid, amongothers.

“Adjuvants” as used herein, can include aluminum hydroxide and aluminumphosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc.,Cambridge Mass.), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham,Ala.), water-in-oil emulsion, oil-in-water emulsion,water-in-oil-in-water emulsion. The emulsion can be based in particularon light liquid paraffin oil (European Pharmacopea type); isoprenoid oilsuch as squalane or squalene oil resulting from the oligomerization ofalkenes, in particular of isobutene or decene; esters of acids or ofalcohols containing a linear alkyl group, more particularly plant oils,ethyl oleate, propylene glycol di-(caprylate/caprate), glyceryltri-(caprylate/caprate) or propylene glycol dioleate; esters of branchedfatty acids or alcohols, in particular isostearic acid esters. The oilis used in combination with emulsifiers to form the emulsion. Theemulsifiers are preferably nonionic surfactants, in particular esters ofsorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, ofpolyglycerol, of propylene glycol and of oleic, isostearic, ricinoleicor hydroxystearic acid, which are optionally ethoxylated, andpolyoxypropylene-polyoxyethylene copolymer blocks, in particular thePluronic products, especially L121. See Hunter et al., The Theory andPractical Application of Adjuvants (Ed. Stewart-Tull, D. E. S.).JohnWiley and Sons, NY, pp 51-94 (1995) and Todd et al., Vaccine15:564-570 (1997).

For example, it is possible to use the SPT emulsion described on page147 of “Vaccine Design, The Subunit and Adjuvant Approach” edited by M.Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59described on page 183 of this same book.

A further instance of an adjuvant is a compound chosen from the polymersof acrylic or methacrylic acid and the copolymers of maleic anhydrideand alkenyl derivative. Advantageous adjuvant compounds are the polymersof acrylic or methacrylic acid which are cross-linked, especially withpolyalkenyl ethers of sugars or polyalcohols. These compounds are knownby the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Personsskilled in the art can also refer to U.S. Pat. No. 2,909,462 whichdescribes such acrylic polymers cross-linked with a polyhydroxylatedcompound having at least 3 hydroxyl groups, preferably not more than 8,the hydrogen atoms of at least three hydroxyls being replaced byunsaturated aliphatic radicals having at least 2 carbon atoms. Thepreferred radicals are those containing from 2 to 4 carbon atoms, e.g.vinyls, allyls and other ethylenically unsaturated groups. Theunsaturated radicals may themselves contain other substituents, such asmethyl. The products sold under the name Carbopol; (BF Goodrich, Ohio,USA) are particularly appropriate. They are cross-linked with an allylsucrose or with allyl pentaerythritol. Among them, there may bementioned Carbopol 974P, 934P and 971P. Most preferred is the use ofCarbopol, in particular the use of Carbopol 971P, preferably in amountsof about 500 μg to about 5 mg per dose, even more preferred in an amountof about 750 μg to about 2.5 mg per dose and most preferred in an amountof about 1 mg per dose.

Further suitable adjuvants include, but are not limited to, the RIBIadjuvant system (Ribi Inc.), Block co-polymer (CytRx, Atlanta Ga.),SAF-M (Chiron, Emeryville Calif.), monophosphoryl lipid A, Avridinelipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinantor otherwise), cholera toxin, IMS 1314, or muramyl dipeptide among manyothers.

Preferably, the adjuvant is added in an amount of about 100 μg to about10 mg per dose. Even more preferably, the adjuvant is added in an amountof about 100 μg to about 10 mg per dose. Even more preferably, theadjuvant is added in an amount of about 500 μg to about 5 mg per dose.Even more preferably, the adjuvant is added in an amount of about 750 μgto about 2.5 mg per dose. Most preferably, the adjuvant is added in anamount of about 1 mg per dose.

Additionally, the composition can include one or morepharmaceutical-acceptable carriers. As used herein, “apharmaceutical-acceptable carrier” includes any and all solvents,dispersion media, coatings, stabilizing agents, diluents, preservatives,antibacterial and antifungal agents, isotonic agents, adsorptiondelaying agents, and the like. Most preferably, the composition providedherewith, contains PCV2 ORF2 protein recovered from the supernate of invitro cultured cells, wherein said cells were infected with arecombinant viral vector containing PCV2 ORF2 DNA and expressing PCV2ORF2 protein, and wherein said cell culture was treated with about 2 toabout 8 mM BEI, preferably with about 5 mM BEI to inactivate the viralvector, and an equivalent concentration of a neutralization agent,preferably sodium thiosulfate solution to a final concentration of about2 to about 8 mM, preferably of about 5 mM.

The present invention also relates to an immunogenic composition thatcomprises i) any of the PCV2 ORF2 proteins described above, preferablyin concentrations described above, ii) at least a portion of the viralvector expressing said PCV2 ORF2 protein, iii) a portion of the cellculture, iv) an inactivating agent to inactivate the recombinant viralvector preferably BEI, and v) an neutralization agent to stop theinactivation mediated by the inactivating agent, preferably sodiumthiosulfate in equivalent amounts to BEI; and vi) a suitable adjuvant,preferably Carbopol 971 in amounts described above; wherein about 90% ofthe components i) to iii) have a size smaller than 1 μm. According to afurther aspect, this immunogenic composition further comprises apharmaceutical acceptable salt, preferably a phosphate salt inphysiologically acceptable concentrations. Preferably, the pH of saidimmunogenic composition is adjusted to a physiological pH, meaningbetween about 6.5 and 7.5.

The immunogenic composition as used herein also refers to a compositionthat comprises per one ml i) at least 1.6 μg of PCV2 ORF2 proteindescribed above, ii) at least a portion of baculovirus expressing saidPCV2 ORF2 protein iii) a portion of the cell culture, iv) about 2 to 8mM BEI, v) sodium thiosulfate in equivalent amounts to BEI; and vi)about 1 mg Carbopol 971, and vii) phosphate salt in a physiologicallyacceptable concentration; wherein about 90% of the components i) to iii)have a size smaller than 1 μm and the pH of said immunogenic compositionis adjusted to about 6.5 to 7.5.

The immunogenic compositions can further include one or more otherimmunomodulatory agents such as, e.g., interleukins, interferons, orother cytokines. The immunogenic compositions can also includeGentamicin and Merthiolate. While the amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan, the present inventioncontemplates compositions comprising from about 50 μg to about 2000 μgof adjuvant and preferably about 250 μg/ml dose of the vaccinecomposition. Thus, the immunogenic composition as used herein alsorefers to a composition that comprises from about 1 ug/ml to about 60μg/ml of antibiotics, and more preferably less than about 30 μg/ml ofantibiotics.

The immunogenic composition as used herein also refers to a compositionthat comprises i) any of the PCV2 ORF2 proteins described above,preferably in concentrations described above, ii) at least a portion ofthe viral vector expressing said PCV2 ORF2 protein, iii) a portion ofthe cell culture, iv) an inactivating agent to inactivate therecombinant viral vector preferably BEI, and v) an neutralization agentto stop the inactivation mediated by the inactivating agent, preferablysodium thiosulfate in equivalent amounts to BEI; vi) a suitableadjuvant, preferably Carbopol 971 in amounts described above; vii) apharmaceutical acceptable concentration of a saline buffer, preferablyof a phosphate salt, and viii) an anti-microbiological active agent;wherein about 90% of the components i) to iii) have a size smaller than1 μm.

It has been surprisingly found, that the immunogenic compositioncomprising the PCV2 ORF2 protein was highly stable over a period of 24months. It has also been found the immunogenic compositions are veryeffective in reducing the clinical symptoms associated with PCV2infections. It was also discovered, that the immunogenic compositionscomprising the recombinant baculovirus expressed PCV2 ORF2 protein asdescribed above, are surprisingly more effective than an immunogeniccomposition comprising the whole PCV2 virus in an inactivated form, orisolated viral PCV2 ORF2 antigen. In particular, it has beensurprisingly found, that the recombinant baculovirus expressed PCV2 ORF2protein is effective in very low concentrations, which means inconcentrations up to 0.25 μg/dose. This unexpected high immunogenicpotential of the PCV2 ORF2 protein is increased by Carbopol. Examples 1to 3 disclose in detail the production of PCV2 ORF2 comprisingimmunogenic compositions.

The immunogenic composition as used herein also refers to IngelVac®CircoFLEX™, (Boehringer Ingelheim Vetmedica, Inc., St Joseph, Mo., USA),CircoVac® (Merial SAS, Lyon, France), CircoVent (Intervet Inc.,Millsboro, Del., USA), or Suvaxyn PCV-2 One Dose® (Fort Dodge AnimalHealth, Kansas City, Kans., USA).

Administration of the Immunogenic Composition

The composition according to the invention may be applied intradermally,intratracheally, or intravaginally. The composition preferably may beapplied intramuscularly or intranasally, most preferablyintramuscularly. In an animal body, it can prove advantageous to applythe pharmaceutical compositions as described above via an intravenous orby direct injection into target tissues. For systemic application, theintravenous, intravascular, intramuscular, intranasal, intraarterial,intraperitoneal, oral, or intrathecal routes are preferred. A more localapplication can be effected subcutaneously, intradermally,intracutaneously, intracardially, intralobally, intramedullary,intrapulmonarily or directly in or near the tissue to be treated(connective-, bone-, muscle-, nerve-, epithelial tissue). Depending onthe desired duration and effectiveness of the treatment, thecompositions according to the invention may be administered once orseveral times, also intermittently, for instance on a daily basis forseveral days, weeks or months and in different dosages.

Preferably, at least one dose of the immunogenic compositions asdescribed above is intramuscularly administered to the subject in needthereof. According to a further aspect, the PCV-2 antigen or theimmunogenic composition comprising any such PCV-2 antigen as describedabove is formulated and administered in one (1) mL per dose. Thus,according to a further aspect, the present invention also relates to a 1ml immunogenic composition, comprising PCV-2 antigen as describedherein, for reducing or lessening lymphadenopathy, lymphoid depletionand/or multinucleated/giant histiocytes in pigs infected with PCV2.

According to a further aspect, according to a further aspect, thepresent invention also relates to a 1 ml immunogenic composition,comprising PCV-2 antigen as described herein, for reducing or lesseninglymphadenopathy in combination with one or a multiple of the followingsymptoms in pigs: (1) interstitial pneumonia with interlobular edema,(2) cutaneous pallor or icterus, (3) mottled atrophic livers, (4)gastric ulcers, (5) nephritis and (6) reproductive disorders, e.g.abortion, stillbirths, mummies.

According to a further aspect, at least one further administration of atleast one dose of the immunogenic composition as described above isgiven to a subject in need thereof, wherein the second or any furtheradministration is given at least 14 days beyond the initial or anyformer administrations. Preferably, the immunogenic composition isadministered with an immune stimulant. Preferably, said immune stimulantis given at least twice. Preferably, at least 3 days, more preferably atleast 5 days, even more preferably at least 7 days are in between thefirst and the second or any further administration of the immunestimulant. Preferably, the immune stimulant is given at least 10 days,preferably 15 days, even more preferably 20, even more preferably atleast 22 days beyond the initial administration of the immunogeniccomposition provided herein. A preferred immune stimulant is, forexample, keyhole limpet hemocyanin (KLH), preferably emulsified withincomplete Freund's adjuvant (KLH/ICFA). However, it is herewithunderstood, that any other immune stimulant known to a person skilled inthe art can also be used. The term “immune stimulant” as used herein,means any agent or composition that can trigger the immune response,preferably without initiating or increasing a specific immune response,for example the immune response against a specific pathogen. It isfurther instructed to administer the immune stimulant in a suitabledose.

Moreover, it has also been surprisingly found that the immunogenicpotential of the immunogenic compositions used herein, preferably thosethat comprise recombinant baculovirus expressed PCV2 ORF2 protein, evenmore preferably in combination with Carbopol, can be further confirmedby the administration of the IngelVac PRRS MLV vaccine (see Example 5).PCV2 clinical signs and disease manifestations are greatly magnifiedwhen PRRS infection is present. However, the immunogenic compositionsand vaccination strategies as provided herewith lessened this effectgreatly, and more than expected. In other words, an unexpectedsynergistic effect was observed when animals, preferably piglets weretreated with any of the PCV2 ORF2 immunogenic compositions, as providedherewith, and the IngelVac PRRS MLV vaccine (Boehringer Ingelheim).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic flow diagram of a preferred construction of PCV2ORF2 recombinant baculovirus; and

FIGS. 2 a and 2 b are each schematic flow diagrams of how to produce oneof the compositions used in accordance with the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples set forth preferred materials and procedures inaccordance with the present invention. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. It is to be understood,however, that these examples are provided by way of illustration only,and nothing therein should be deemed a limitation upon the overall scopeof the invention.

Example 1

This example compares the relative yields of ORF2 using methods of thepresent invention with methods that are known in the prior art. Four1000 mL spinner flasks were each seeded with approximately 1.0×10⁶ Sf+cells/ml in 300 mL of insect serum free media, Excell 420 (JRHBiosciences, Inc., Lenexa, Kans.). The master cell culture is identifiedas SF+ (Spodoptera frugiperda) Master Cell Stock, passage 19,Lot#N112-095W. The cells used to generate the SF+Master Cell Stock wereobtained from Protein Sciences Corporation, Inc., Meriden, Conn. The SF+cell line for this example was confined between passages 19 and 59.Other passages will work for purposes of the present invention, but inorder to scale the process up for large scale production, at least 19passages will probably be necessary and passages beyond 59 may have aneffect on expression, although this was not investigated. In moredetail, the initial SF+ cell cultures from liquid nitrogen storage weregrown in Excell 420 media in suspension in sterile spinner flasks withconstant agitation. The cultures were grown in 100 mL to 250 mL spinnerflasks with 25 to 150 mL of Excell 420 serum-free media. When the cellshad multiplied to a cell density of 1.0-8.0×10⁶ cells/mL, they weresplit to new vessels with a planting density of 0.5-1.5×10⁶ cells/mL.Subsequent expansion cultures were grown in spinner flasks up to 36liters in size or in stainless steel bioreactors of up to 300 liters fora period of 2-7 days at 25-29° C.

After seeding, the flasks were incubated at 27° C. for four hours.Subsequently, each flask was seeded with a recombinant baculoviruscontaining the PCV2 ORF2 gene (SEQ ID NO: 4). The recombinantbaculovirus containing the PCV2 ORF2 gene was generated as follows: thePCV2 ORF2 gene from a North American strain of PCV2 was PCR amplified tocontain a 5′ Kozak's sequence (SEQ ID NO: 1) and a 3′ EcoR1 site (SEQ IDNO: 2), and cloned into the pGEM-T-Easy vector (Promega, Madison, Wis.).Then, it was subsequently excised and subcloned into the transfer vectorpVL1392 (BD Biosciences Pharmingen, San Diego, Calif.). The subclonedportion is represented herein as SEQ ID NO: 7. The pVL1392 plasmidcontaining the PCV2 ORF2 gene was designated N47-064Y and thenco-transfected with BaculoGold® (BD Biosciences Pharmingen) baculovirusDNA into Sf+ insect cells (Protein Sciences, Meriden, Conn.) to generatethe recombinant baculovirus containing the PCV2 ORF2 gene. The newconstruct is provided herein as SEQ ID NO: 8. The recombinantbaculovirus containing the PCV2 ORF2 gene was plaque-purified and MasterSeed Virus (MSV) was propagated on the SF+ cell line, aliquotted, andstored at −70° C. The MSV was positively identified as PCV2 ORF2baculovirus by PCR-RFLP using baculovirus specific primers. Insect cellsinfected with PCV2 ORF2 baculovirus to generate MSV or Working SeedVirus express PCV2 ORF2 antigen as detected by polyclonal serum ormonoclonal antibodies in an indirect fluorescent antibody assay.Additionally, the identity of the PCV2 ORF2 baculovirus was confirmed byN-terminal amino acid sequencing. The PCV2 ORF2 baculovirus MSV was alsotested for purity in accordance with 9 C.F.R. 113.27 (c), 113.28, and113.55. Each recombinant baculovirus seeded into the spinner flasks hadvarying multiplicities of infection (MOIs). Flask 1 was seeded with 7.52mL of 0.088 MOI seed; flask 2 was seeded with 3.01 mL of 0.36 MOI seed;flask 3 was seeded with 1.5 mL of 0.18 MOI seed; and flask 4 was seededwith 0.75 mL of 0.09 MOI seed. A schematic flow diagram illustrating thebasic steps used to construct a PCV2 ORF2 recombinant baculovirus isprovided herein as FIG. 1.

After being seeded with the baculovirus, the flasks were then incubatedat 27±2° C. for 7 days and were also agitated at 100 rpm during thattime. The flasks used ventilated caps to allow for air flow. Samplesfrom each flask were taken every 24 hours for the next 7 days. Afterextraction, each sample was centrifuged, and both the pellet and thesupernatant were separated and then microfiltered through a 0.45-1.0 μmpore size membrane.

The resulting samples then had the amount of ORF2 present within themquantified via an ELISA assay. The ELISA assay was conducted withcapture antibody Swine anti-PCV2 Pab IgG Prot. G purified (diluted 1:250in PBS) diluted to 1:6000 in 0.05M Carbonate buffer (pH 9.6). 100 μL ofthe antibody was then placed in the wells of the mictrotiter plate,sealed, and incubated overnight at 37° C. The plate was then washedthree times with a wash solution which comprised 0.5 mL of Tween 20(Sigma, St. Louis, Mo.), 100 mL of 10× D-PBS (Gibco Invitrogen,Carlsbad, Calif.) and 899.5 mL of distilled water. Subsequently, 250 μLof a blocking solution (5 g Carnation Non-fat dry milk (Nestle,Glendale, Calif.) in 10 mL of D-PBS QS to 100 mL with distilled water)was added to each of the wells. The next step was to wash the test plateand then add pre-diluted antigen. The pre-diluted antigen was producedby adding 200 μL of diluent solution (0.5 mL Tween 20 in 999.5 mL D-PBS)to each of the wells on a dilution plate. The sample was then diluted ata 1:240 ratio and a 1:480 ratio, and 100 μL of each of these dilutedsamples was then added to one of the top wells on the dilution plate(i.e. one top well received 100 μL of the 1:240 dilution and the otherreceived 100 μL of the 1:480 dilution). Serial dilutions were then donefor the remainder of the plate by removing 100 μL form each successivewell and transferring it to the next well on the plate. Each well wasmixed prior to doing the next transfer. The test plate washing includedwashing the plate three times with the wash buffer. The plate was thensealed and incubated for an hour at 37° C. before being washed threemore times with the wash buffer. The detection antibody used wasmonoclonal antibody to PCV ORF2. It was diluted to 1:300 in diluentsolution, and 100 μL of the diluted detection antibody was then added tothe wells. The plate was then sealed and incubated for an hour at 37° C.before being washed three times with the wash buffer. Conjugate diluentwas then prepared by adding normal rabbit serum (Jackson Immunoresearch,West Grove, Pa.) to the diluent solution to 1% concentration. Conjugateantibody Goat anti-mouse (H+1)-HRP (Jackson Immunoresearch) was dilutedin the conjugate diluent to 1:10,000. 100 μL of the diluted conjugateantibody was then added to each of the wells. The plate was then sealedand incubated for 45 minutes at 37° C. before being washed three timeswith the wash buffer. 100 μL of substrate (TMB Peroxidase Substrate,Kirkgaard and Perry Laboratories (KPL), Gaithersberg, Md.), mixed withan equal volume of Peroxidase Substrate B (KPL) was added to each of thewells. The plate was incubated at room temperature for 15 minutes. 100μL of 1N HCL solution was then added to all of the wells to stop thereaction. The plate was then run through an ELISA reader. The results ofthis assay are provided in Table 1 below:

TABLE 1 ORF2 Day Flask in pellet (μg) ORF2 in supernatant (μg) 3 1 47.5312 3 2 57.46 22 3 3 53.44 14 3 4 58.64 12 4 1 43.01 44 4 2 65.61 62 4 370.56 32 4 4 64.97 24 5 1 31.74 100 5 2 34.93 142 5 3 47.84 90 5 4 55.1486 6 1 14.7 158 6 2 18.13 182 6 3 34.78 140 6 4 36.88 146 7 1 6.54 176 72 12.09 190 7 3 15.84 158 7 4 15.19 152

These results indicate that when the incubation time is extended,expression of ORF2 into the supernatant of the centrifuged cells andmedia is greater than expression in the pellet of the centrifuged cellsand media. Accordingly, allowing the ORF2 expression to proceed for atleast 5 days and recovering it in the supernate rather than allowingexpression to proceed for less than 5 days and recovering ORF2 from thecells, provides a great increase in ORF2 yields, and a significantimprovement over prior methods.

Example 2

This example provides data as to the efficacy of the invention claimedherein. A 1000 mL spinner flask was seeded with approximately 1.0×10⁶Sf+ cells/ml in 300 mL of Excell 420 media. The flask was then incubatedat 27° C. and agitated at 100 rpm. Subsequently, the flask was seededwith 10 mL of PCV2 ORF2/Bac p+6 (the recombinant baculovirus containingthe PCV2 ORF2 gene passaged 6 additional times in the Sf9 insect cells)virus seed with a 0.1 MOI after 24 hours of incubation.

The flask was then incubated at 27° C. for a total of 6 days. Afterincubation, the flask was then centrifuged and three samples of theresulting supernatant were harvested and inactivated. The supernatantwas inactivated by bringing its temperature to 37±2° C. To the firstsample, a 0.4M solution of 2-bromoethyleneamine hydrobromide which hadbeen cyclized to 0.2M binary ethlylenimine (BEI) in 0.3N NaOH was addedto the supernatant to give a final concentration of BEI of 5 mM. To thesecond sample, 10 mM BEI was added to the supernatant. To the thirdsample, no BEI was added to the supernatant. The samples were thenstirred continuously for 48 hrs. A 1.0 M sodium thiosulfate solution togive a final minimum concentration of 5 mM was added to neutralize anyresidual BEI. The quantity of ORF2 in each sample was then quantifiedusing the same ELISA assay procedure as described in Example 1. Theresults of this may be seen in Table 2 below:

TABLE 2 Sample ORF2 in supernatant (μg) 1 78.71 2 68.75 3 83.33

This example demonstrates that neutralization with BEI does not removeor degrade significant amounts of the recombinant PCV2 ORF2 proteinproduct. This is evidenced by the fact that there is no large loss ofORF2 in the supernatant from the BEI or elevated temperatures. Those ofskill in the art will recognize that the recovered ORF2 is a stableprotein product.

Example 3

This example demonstrates that the present invention is scalable fromsmall scale production of recombinant PCV2 ORF2 to large scaleproduction of recombinant PCV2 ORF2. 5.0×10⁵ cells/ml of SF+ cells/ml in700 mL of ExCell 420 media was planted in a 20000 mL ApplikonBioreactor. The media and cells were then incubated at 27° C. andagitated at 100 RPM for the next 68 hours. At the 68^(th) hour, 41.3 mLof PCV2 ORF2 Baculovirus MSV+3 was added to 7000 mL of ExCell 420medium. The resultant mixture was then added to the bioreactor. For thenext seven days, the mixture was incubated at 27° C. and agitated at 100RPM. Samples from the bioreactor were extracted every 24 hours beginningat day 4, post-infection, and each sample was centrifuged. Thesupernatant of the samples were preserved and the amount of ORF2 wasthen quantified using SDS-PAGE densitometry. The results of this can beseen in Table 3 below:

TABLE 3 Day after infection: ORF2 in supernatant (μg/mL) 4 29.33 5 41.336 31.33 7 60.67

Example 4

This example tests the efficacy of seven PCV2 candidate vaccines andfurther defines efficacy parameters following exposure to a virulentstrain of PCV2. One hundred and eight (108) cesarean derived colostrumdeprived (CDCD) piglets, 9-14 days of age, were randomly divided into 9groups of equal size. Table 4 sets forth the General Study Design forthis Example.

TABLE 4 General Study Design Challenged KLH/ICFA with on Day VirulentNo. Of Day of 21 and PCV2 on Necropsy Group Pigs Treatment Treatment Day27 Day 24 on Day 49 1 12 PCV2 Vaccine No. 0 + + + 1 - (vORF2 16 μg) 2 12PCV2 Vaccine No. 0 + + + 2 - (vORF2 8 μg) 3 12 PCV2 Vaccine No. 0 + + +3 - (vORF2 4 μg) 4 12 PCV2 Vaccine No. 0 + + + 4 - (rORF2 16 μg) 5 12PCV2 Vaccine No. 0 + + + 5 - (rORF2 8 μg) 6 12 PCV2 Vaccine No. 0 + + +6 - (rORF2 4 μg) 7 12 PCV2 Vaccine No. 0 + + + 7 - (Killed whole cellvirus) 8 12 None - Challenge N/A + + + Controls 9 12 None - Strict N/A +− + Negative Control Group vORF2 = isolated viral ORF2; rORF2 =recombinant baculovirus expressed ORF2; killed whole cell virus = PCV2virus grown in suitable cell culture

Seven of the groups (Groups 1-7) received doses of PCV2 ORF2polypeptide, one of the groups acted as a challenge control and receivedno PCV2 ORF2, and another group acted as the strict negative controlgroup and also received no PCV2 ORF2. On Day 0, Groups 1 through 7 weretreated with assigned vaccines. Piglets in Group 7 were given a boostertreatment on Day 14. Piglets were observed for adverse events andinjection site reactions following vaccination and on Day 19, pigletswere moved to the second study site. At the second study site, Groups1-8 were group housed in one building while Group 9 was housed in aseparate building. All pigs received keyhole limpet hemocyanin(KLH)/incomplete Freund's adjuvant (ICFA) on Days 21 and 27 and on Day24, Groups 1-8 were challenged with a virulent PCV2.

Pre- and post-challenge, blood samples were collected for PCV2 serology.Post-challenge, body weight data for determination of average dailyweight gain (ADWG), and clinical symptoms, as well as nasal swab samplesto determine nasal shedding of PCV2, were collected. On Day 49, allsurviving pigs were necropsied, lungs were scored for lesions, andselected tissues were preserved in formalin for Immunohistochemistry(IHC) testing at a later date.

Materials and Methods

This was a partially blinded vaccination-challenge feasibility studyconducted in CDCD pigs, 9 to 14 days of age on Day 0. To be included inthe study, PCV2 IFA titers of sows were ≦1:1000. Additionally, theserologic status of sows were from a known PRRS-negative herd.Twenty-eight (28) sows were tested for PCV2 serological status. Fourteen(14) sows had a PCV2 titer of ≦1000 and were transferred to the firststudy site. One hundred ten (110) piglets were delivered by cesareansection surgeries and were available for this study on Day −4. On Day−3, 108 CDCD pigs at the first study site were weighed, identified withear tags, blocked by weight and randomly assigned to I of 9 groups, asset forth above in table 4. If any test animal meeting the inclusioncriteria was enrolled in the study and was later excluded for anyreason, the Investigator and Monitor consulted in order to determine theuse of data collected from the animal in the final analysis. The date ofwhich enrolled piglets were excluded and the reason for exclusion wasdocumented. Initially, no sows were excluded. A total of 108 of anavailable 110 pigs were randomly assigned to one of 9 groups on Day −3.The two smallest pigs (Nos. 17 and 19) were not assigned to a group andwere available as extras, if needed. During the course of the study,several animals were removed. Pig 82 (Group 9) on Day −1, Pig No. 56(Group 6) on Day 3, Pig No. 53 (Group 9) on Day 4, Pig No. 28 (Group 8)on Day 8, Pig No. 69 (Group 8) on Day 7, and Pig No. 93 (Group 4) on Day9, were each found dead prior to challenge. These six pigs were notincluded in the final study results. Pig no 17 (one of the extra pigs)was assigned to Group 9. The remaining extra pig, No. 19, was excludedfrom the study.

The formulations given to each of the groups were as follows: Group 1was designed to administer 1 ml of viral ORF2 (vORF2) containing 16 μgORF2/ml. This was done by mixing 10.24 ml of viral ORF2 (256 μg/25μg/ml=10.24 ml vORF2) with 3.2 ml of 0.5% Carbopol and 2.56 ml ofphosphate buffered saline at a pH of 7.4. This produced 16 ml offormulation for group 1. Group 2 was designed to administer 1 ml ofvORF2 containing 8 μg vORF2/ml. This was done by mixing 5.12 ml of vORF2(128 μg/25 μg/ml=5.12 ml vORF2) with 3.2 ml of 0.5% Carbopol and 7.68 mlof phosphate buffered saline at a pH of 7.4. This produced 16 ml offormulation for group 2. Group 3 was designed to administer 1 ml ofvORF2 containing 4 μg vORF2/ml. This was done by mixing 2.56 ml of vORF2(64 μg/25 μg/ml=2.56 ml vORF2) with 3.2 ml of 0.5% Carbopol and 10.24 mlof phosphate buffered saline at a pH of 7.4. This produced 16 ml offormulation for group 3. Group 4 was designed to administer 1 ml ofrecombinant ORF2 (rORF2) containing 16 μg rORF2/ml. This was done bymixing 2.23 ml of rORF2 (512 μg/230 μg/ml=2.23 ml rORF2) with 6.4 ml of0.5% Carbopol and 23.37 ml of phosphate buffered saline at a pH of 7.4.This produced 32 ml of formulation for group 4. Group 5 was designed toadminister 1 ml of rORF2 containing 8 μg rORF2/ml. This was done bymixing 1.11 ml of rORF2 (256 μg/230 μg/1 ml=1.11 ml rORF2) with 6.4 mlof 0.5% Carbopol and 24.49 ml of phosphate-buffered saline at a pH of7.4. This produced 32 ml of formulation for group 5. Group 6 wasdesigned to administer 1 ml of rORF2 containing 8 μg rORF2/ml. This wasdone by mixing 0.56 ml of rORF2 (128 μg/230 μg/ml=0.56 ml rORF2) with6.4 ml of 0.5% Carbopol and 25.04 ml of phosphate buffered saline at apH of 7.4. This produced 32 ml of formulation for group 6. Group 7 wasdesigned to administer 2 ml of PCV2 whole killed cell vaccine (PCV2 KV)containing the MAX PCV2 KV. This was done by mixing 56 ml of PCV2 KVwith 14 ml of 0.5% Carbopol. This produced 70 ml of formulation forgroup 7. Finally group 8 was designed to administer KLH at 0.5 μg/ml or1.0 μg/ml per 2 ml dose. This was done by mixing 40.71 ml KLH (7.0 μgprotein/ml at 0.5 μg/ml=570 ml (7.0 μg/ml)(x)=(0.5)(570 ml)), 244.29 mlphosphate buffered saline at a pH of 7.4, and 285 ml Freunds adjuvant.Table 5 describes the time frames for the key activities of thisExample.

TABLE 5 Study Activities Study Day Study Activity −4, 0 to Generalobservations for overall health and clinical symptoms 49 −3 Weighed;Randomized to groups; Collected blood samples from all pigs  0 Healthexamination; Administered IVP Nos. 1-7 to Groups 1-7, respectively 0-7Observed pigs for injection site reactions 14 Boostered Group 7 withPCV2 Vaccine No. 7; Blood samples from all pigs 14-21 Observed Group 7for injection site reactions 16-19 Treated all pigs with antibiotics(data missing) 19 Pigs transported from the first test site to a secondtest site 21 Treated Groups 1-9 with KLH/ICFA 24 Collected blood andnasal swab samples from all pigs; Weighed all pigs; Challenged Groups1-8 with PCV2 challenge material 25, 27, Collected nasal swab samplesfrom all pigs 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 27 Treated Groups1-9 with KLH/ICFA 31 Collected blood samples from all pigs 49 Collectedblood and nasal swab samples from all pigs; Weighed all pigs; Necropsyall pigs; Gross lesions noted with emphasis placed on icterus andgastric ulcers; Lungs evaluated for lesions; Fresh and formalin fixedtissue samples saved; In- life phase of the study completed

Following completion of the in-life phase of the study, formalin fixedtissues w % ere examined by Immunohistochemistry (IHC) for detection ofPCV2 antigen by a pathologist, blood samples were evaluated for PCV2serology, nasal swab samples were evaluated for PCV2 shedding, andaverage daily weight gain (ADWG) was determined from Day 24 to Day 49.

Animals were housed at the first study site in individual cages in fiverooms from birth to approximately 11 days of age (approximately Day 0 ofthe study). Each room was identical in layout and consisted of stackedindividual stainless steel cages with heated and filtered air suppliedseparately to each isolation unit. Each room had separate heat andventilation, thereby preventing cross-contamination of air betweenrooms. Animals were housed in two different buildings at the secondstudy site. Group 9 (The Strict negative control group) was housedseparately in a converted finisher building and Groups 1-8 were housedin converted nursery building. Each group was housed in a separate pen(11-12 pigs per pen) and each pen provided approximately 3.0 square feetper pig. Each pen was on an elevated deck with plastic slatted floors. Apit below the pens served as a holding tank for excrement and waste.Each building had its own separate heating and ventilation systems, withlittle likelihood of cross-contamination of air between buildings.

At the first study site, piglets were fed a specially formulated milkration from birth to approximately 3 weeks of age. All piglets wereconsuming solid, special mixed ration by Day 19 (approximately 4½ weeksof age). At the second study site, all piglets were fed a customnon-medicated commercial mix ration appropriate for their age andweight, ad libitum. Water at both study sites was also available adlibitum.

All test pigs were treated with Vitamin E on Day −2, with ironinjections on Day −1 and with NAXCEL® (1.0 mL, IM, in alternating hams)on Days 16, 17, 18 and 19. In addition, Pig No. 52 (Group 9) was treatedwith an iron injection on Day 3, Pig 45 (Group 6) was treated with aniron injection on Day 11, Pig No. 69 (Group 8) was treated with NAXCEL®on Day 6, Pig No. 74 (Group 3) was treated with dexamethazone andpenicillin on Day 14, and Pig No. 51 (Group 1) was treated withdexamethazone and penicillin on Day 13 and with NAXCEL® on Day 14 forvarious health reasons.

While at both study sites, pigs were under veterinary care. Animalhealth examinations were conducted on Day 0 and were recorded on theHealth Examination Record Form. All animals were in good health andnutritional status before vaccination as determined by observation onDay 0. All test animals were observed to be in good health andnutritional status prior to challenge. Carcasses and tissues weredisposed of by rendering. Final disposition of study animals was recordson the Animal Disposition Record.

On Day 0, pigs assigned to Groups 1-6 received 1.0 mL of PCV2 Vaccines1-6, respectively, IM in the left neck region using a sterile 3.0 mLLuer-lock syringe and a sterile 20 g×½″ needle. Pigs assigned to Group 7received 2.0 mL of PCV2 Vaccine No. 7 IM in the left neck region using asterile 3.0 mL Luer-lock syringe and a sterile 20 g×2″ needle. On Day14, pigs assigned to Group 7 received 2.0 mL of PCV2 Vaccine No. 7 IM inthe right neck region using a sterile 3.0 mL Luer-lock syringe and asterile 20 g×½″ needle.

On Day 21 all test pigs received 2.0 mL of KLH/ICFA IM in the right hamregion using a sterile 3.0 mL Luer-lock syringe and a sterile 20 g×1″needle. On Day 27 all test pigs received 2.0 mL of KLH/ICFA in the leftham region using a sterile 3.0 mL Luer-lock syringe and a sterile 20g×1″ needle.

On Day 24, pigs assigned to Groups 1-8 received 1.0 mL of PCV2 ISUVDLchallenge material (5.11 log₁₀ TCID₅₀/mL) IM in the left neck regionusing a sterile 3.0 mL Luer-lock syringe and a sterile 20 g×1″ needle.An additional 1.0 mL of the same material was administered IN to eachpig (0.5 mL per nostril) using a sterile 3.0 mL Luer-lock syringe andnasal canula.

Test pigs were observed daily for overall health and adverse events onDay −4 and from Day 0 to Day 19. Observations were recorded on theClinical Observation Record. All test pigs were observed from Day 0 toDay 7, and Group 7 was further observed from Day 14 to 21, for injectionsite reactions. Average daily weight gain was determined by weighingeach pig on a calibrated scale on Days −3, 24 and 49, or on the day thata pig was found dead after challenge. Body weights were recorded on theBody Weight Form. Day −3 body weights were utilized to block pigs priorto randomization. Day 24 and Day 49 weight data was utilized todetermine the average daily weight gain (ADWG) for each pig during thesetime points. For pigs that died after challenge and before Day 49, theADWG was adjusted to represent the ADWG from Day 24 to the day of death.

In order to determine PCV2 serology, venous whole blood was collectedfrom each piglet from the orbital venous sinus on Days −3 and 14. Foreach piglet, blood was collected from the orbital venous sinus byinserting a sterile capillary tube into the medial canthus of one of theeyes and draining approximately 3.0 mL of whole blood into a 4.0 mLSerum Separator Tube (SST). On Days 24, 31, and 49, venous whole bloodfrom each pig was collected from the anterior vena cava using a sterile18 g×1½′ Vacutainer needle (Becton Dickinson and Company, FranklinLakes, N.J.), a Vacutainer needle holder and a 13 mL SST. Bloodcollections at each time point were recorded on the Sample CollectionRecord. Blood in each SST was allowed to clot, each SST was then spundown and the serum harvested. Harvested serum was transferred to asterile snap tube and stored at −70±10° C. until tested at a later date.Serum samples were tested for the presence of PCV2 antibodies byBIVI-R&D personnel.

Pigs were observed once daily from Day 20 to Day 49 for clinicalsymptoms and clinical observations were recorded on the ClinicalObservation Record.

To test for PCV2 nasal shedding, on Days 24, 25, and then every otherodd numbered study day up to and including Day 49, a sterile dacron swabwas inserted intra nasally into either the left or right nostril of eachpig (one swab per pig) as aseptically as possible, swished around for afew seconds and then removed. Each swab was then placed into a singlesterile snap-cap tube containing 1.0 mL of EMEM media with 2% IFBS, 500units/mL of Penicillin, 500 μg/1 mL of Streptomycin and 2.5 μg/mL ofFungizone. The swab was broken off in the tube, and the snap tube wassealed and appropriately labeled with animal number, study number, dateof collection, study day and “nasal swab.” Sealed snap tubes were storedat −40±10° C. until transported overnight on ice to BIVI-St. Joseph.Nasal swab collections were recorded on the Nasal Swab Sample CollectionForm. BIVI-R&D conducted quantitative virus isolation (VI) testing forPCV2 on nasal swab samples. The results were expressed in log₁₀ values.A value of 1.3 logs or less was considered negative and any valuegreater than 1.3 logs was considered positive.

Pigs that died (Nos. 28, 52, 56, 69, 82, and 93) at the first study sitewere necropsied to the level necessary to determine a diagnosis. Grosslesions were recorded and no tissues were retained from these pigs. Atthe second study site, pigs that died prior to Day 49 (Nos. 45, 23, 58,35), pigs found dead on Day 49 prior to euthanasia (Nos. 2, 43), andpigs euthanized on Day 49 were necropsied. Any gross lesions were notedand the percentages of lung lobes with lesions were recorded on theNecropsy Report Form.

From each of the 103 pigs necropsied at the second study site, a tissuesample of tonsil, lung, heart, liver, mesenteric lymph node, kidney andinguinal lymph node was placed into a single container with buffered 10%formalin; while another tissue sample from the same aforementionedorgans was placed into a Whirl-pak (M-Tech Diagnostics Ltd., Thelwall,UK) and each Whirl-pak was placed on ice. Each container was properlylabeled. Sample collections were recorded on the Necropsy Report Form.Afterwards, formalin-fixed tissue samples and a Diagnostic Request Formwere submitted for IHC testing. IHC testing was conducted in accordancewith standard ISU laboratory procedures for receiving samples, sampleand slide preparation, and staining techniques. Fresh tissues inWhirl-paks were shipped with ice packs to the Study Monitor for storage(−70°±10° C.) and possible future use. Formalin-fixed tissues wereexamined by a pathologist for detection of PCV2 by IHC and scored usingthe following scoring system: 0=None; 1=Scant positive staining, fewsites; 2=Moderate positive staining, multiple sites; and 3=Abundantpositive staining, diffuse throughout the tissue. Due to the fact thatthe pathologist could not positively differentiate inguinal LN frommesenteric LN, results for these tissues were simply labeled as LymphNode and the score given the highest score for each of the two tissuesper animal.

Results

Results for this example are given below. It is noted that one pig fromGroup 9 died before Day 0, and 5 more pigs died post-vaccination (1 pigfrom Group 4; 1 pig from Group 6; 2 pigs from Group 8; and 1 pig fromGroup 9). Post-mortem examination indicated all six died due tounderlying infections that were not associated with vaccination or PMWS.Additionally, no adverse events or injection site reactions were notedwith any groups.

Average daily weight gain (ADWG) results are presented below in Table 6.Group 9, the strict negative control group, had the highest ADWG(1.06±0.17 lbs/day), followed by Group 5 (0.94±0.22 lbs/day), whichreceived one dose of 8 μg of rORF2. Group 3, which received one dose of4 μg of vORF2, had the lowest ADWG (0.49±0.21 lbs/day), followed byGroup 7 (0.50±0.15 lbs/day), which received 2 doses of killed vaccine.

TABLE 6 Summary of Group Average Daily Weight Gain (ADWG) ADWG - lbs/day(Day 24 to Day 49) or adjusted for Group Treatment N pigs dead beforeDay 29 1 vORF2 - 16 μg (1 dose) 12 0.87 ± 0.29 lbs/day 2 vORF2 - 8 μg (1dose) 12 0.70 ± 0.32 lbs/day 3 vORF2 - 4 μg (1 dose) 12 0.49 ± 0.21lbs/day 4 rORF2 - 16 μg (1 dose) 11 0.84 ± 0.30 lbs/day 5 rORF2 - 8 μg(1 dose) 12 0.94 ± 0.22 lbs/day 6 rORF2 - 4 μg (1 dose) 11 0.72 ± 0.25lbs/day 7 KV (2 doses) 12 0.50 ± 0.15 lbs/day 8 Challenge Controls 100.76 ± 0.19 lbs/day 9 Strict Negative Controls 11 1.06 ± 0.17 lbs/dayvORF2 = isolated viral ORF2; rORF2 = recombinant baculovirus expressedORF2; killed whole cell virus = PCV2 virus grown in suitable cellculture

PCV2 serology results are presented below in Table 7. All nine groupswere seronegative for PCV2 on Day −3. On Day 14, Groups receiving vORF2vaccines had the highest titers, which ranged from 187.5 to 529.2. Pigsreceiving killed viral vaccine had the next highest titers, followed bythe groups receiving rORF2 vaccines. Groups 8 and 9 remainedseronegative at this time. On Day 24 and Day 31, pigs receiving vORF2vaccines continued to demonstrate a strong serological response,followed closely by the group that received two doses of a killed viralvaccine. Pigs receiving rORF2 vaccines were slower to respondserologically and Groups 8 and 9 continued to remain seronegative. OnDay 49, pigs receiving vORF2 vaccine, 2 doses of the killed viralvaccine and the lowest dose of rORF2 demonstrated the strongestserological responses. Pigs receiving 16 μg and 8 μg of rORF2 vaccineshad slightly higher IFA titers than challenge controls. Group 9 on Day49 demonstrated a strong serological response.

TABLE 7 Summary of Group PCV2 IFA Titers AVERAGE IFA TITER GroupTreatment Day −3 Day 14 Day 24 Day 31** Day 49*** 1 vORF2 - 16 μg (1dose) 50.0 529.2 4400.0 7866.7 11054.5 2 vORF2 - 8 μg (1 dose) 50.0500.0 3466.7 6800.0 10181.8 3 vORF2 - 4 μg (1 dose) 50.0 187.5 1133.35733.3 9333.3 4 rORF2 - 16 μg (1 dose) 50.0 95.5 1550.0 3090.9 8000.0 5rORF2 - 8 μg (1 dose) 50.0 75.0 887.5 2266.7 7416.7 6 rORF2 - 4 μg (1dose) 50.0 50.0 550.0 3118.2 10570.0 7 KV (2 doses) 50.0 204.2 3087.54620.8 8680.0 8 Challenge Controls 50.0 55.0 50.0 50.0 5433.3 9 StrictNegative Controls 50.0 59.1 59.1 54.5 6136.4 vORF2 = isolated viralORF2; rORF2 = recombinant baculovirus expressed ORF2; killed whole cellvirus = PCV2 virus grown in suitable cell culture *For calculationpurposes, a ≦100 IFA titer was designated as a titer of “50”; a ≧6400IFA titer was designated as a titer of “12,800”. **Day of Challenge***Day of Necropsy

The results from the post-challenge clinical observations are presentedbelow in Table 8. This summary of results includes observations forAbnormal Behavior, Abnormal Respiration, Cough and Diarrhea Table 9includes the results from the Summary of Group Overall Incidence ofClinical Symptoms and Table 10 includes results from the Summary ofGroup Mortality Rates Post-challenge. The most common clinical symptomnoted in this study was abnormal behavior, which was scored as mild tosevere lethargy. Pigs receiving the 2 lower doses of vORF2, pigsreceiving 16 μg of rORF2 and pigs receiving 2 doses of KV vaccine hadincidence rates of ≧27.3%. Pigs receiving 8 μg of rORF2 and the strictnegative control group had no abnormal behavior. None of the pigs inthis study demonstrated any abnormal respiration. Coughing was notedfrequently in all groups (0 to 25%), as was diarrhea (0-20%). None ofthe clinical symptoms noted were pathognomic for PMWS.

The overall incidence of clinical symptoms varied between groups. Groupsreceiving any of the vORF2 vaccines, the group receiving 16 μg of rORF2,the group receiving 2 doses of KV vaccine, and the challenge controlgroup had the highest incidence of overall clinical symptoms (≧36.4%).The strict negative control group, the group receiving 8 μg of rORF2 andthe group receiving 4 μg of rORF2 had overall incidence rates ofclinical symptoms of 0%, 8.3% and 9.1%, respectively.

Overall mortality rates between groups varied as well. The groupreceiving 2 doses of KV vaccine had the highest mortality rate (16.7%);while groups that received 4 μg of vORF2, 16 tag of rORF2, or 8 μg ofrORF2 and the strict negative control group all had 0% mortality rates.

TABLE 8 Summary of Group Observations for Abnormal Behavior, AbnormalRespiration, Cough, and Diarrhea Abnormal Abnormal Group Treatment NBehavior¹ Behavior² Cough³ Diarrhea⁴ 1 vORF2 - 16 μg (1 dose) 12 2/12(16.7% 0/12 3/12 (25%) 2/12 (16/7%) (0%) 2 vORF2 - 8 μg (1 dose) 12 4/12(33.3%) 0/12 1/12 (8.3% 1/12 (8.3%) (0%) 3 vORF2 - 4 μg (1 dose) 12 8/12(66.7%) 0/12 2/12 (16.7%) 1/12 (8.3%) (0%) 4 rORF2 - 16 μg (1 dose) 113/11 (27.3%) 0/11 0/11 (0%) 2/11 (18.2%) (0%) 5 rORF2 - 8 μg (1 dose) 120/12 (0%) 0/12 1/12 (8.3%) 0/12 (0%) (0%) 6 rORF2 - 4 μg (1 dose) 111/11 (9.1%) 0/11 0/11 (0%) 0/12 (0%) (0%) 7 KV (2 doses) 12 7/12 (58.3)0/12 0/12 (0%) 1/12 (8.3%) (0%) 8 Challenge Controls 10 1/10 (10%) 0/102/10 (20%) 2/10 (20%) (0%) 9 Strict Negative Controls 11 0/11 (0%) 0/110/11 (0%) 0/11 (0%) (0%) vORF2 = isolated viral ORF2; rORF2 =recombinant baculovirus expressed ORF2; killed whole cell virus = PCV2virus grown in suitable cell culture ¹Total number of pigs in each groupthat demonstrated any abnormal behavior for at least one day ²Totalnumber of pigs in each group that demonstrated any abnormal respirationfor at least one day ³Total number of pigs in each group thatdemonstrated a cough for at least one day ⁴Total number of pigs in eachgroup that demonstrated diarrhea for at least one day

TABLE 9 Summary of Group Overall Incidence of Clinical SymptomsIncidence of pigs with Incidence Group Treatment N Clinical Symptoms¹Rate 1 vORF2 - 16 μg (1 dose) 12 5 41.7% 2 vORF2 - 8 μg (1 dose) 12 541.7% 3 vORF2 - 4 μg (1 dose) 12 8 66.7% 4 rORF2 - 16 μg (1 dose) 11 436.4% 5 rORF2 - 8 μg (1 dose) 12 1  8.3% 6 rORF2 - 4 μg (1 dose) 11 1 9.1% 7 KV (2 doses) 12 7 58.3% 8 Challenge Controls 10 4   40% 9 StrictNegative Controls 11 0   0% vORF2 = isolated viral ORF2; rORF2 =recombinant baculovirus expressed ORF2; killed whole cell virus = PCV2virus grown in suitable cell culture ¹Total number of pigs in each groupthat demonstrated any clinical symptom for at least one day

TABLE 10 Summary of Group Mortality Rates Post-challenge Dead Post-Group Treatment N challenge Mortality Rate 1 vORF2 - 16 μg (1 dose) 12 18.3%   2 vORF2 - 8 μg (1 dose) 12 1 8.3%   3 vORF2 - 4 μg (1 dose) 12 00% 4 rORF2 - 16 μg (1 dose) 11 0 0% 5 rORF2 - 8 μg (1 dose) 12 0 0% 6rORF2 - 4 μg (1 dose) 11 1 9.1%   7 KV (2 doses) 12 2 16.7%   8Challenge Controls 10 1 10%  9 Strict Negative Controls 11 0 0% vORF2 =isolated viral ORF2; rORF2 = recombinant baculovirus expressed ORF2;killed whole cell virus = PCV2 virus grown in suitable cell culture

PCV2 nasal shedding results are presented below in Table 11. Followingchallenge on Day 24, 1 pig in Group 7 began shedding PCV2 on Day 27.None of the other groups experienced shedding until Day 33. The bulk ofnasal shedding was noted from Day 35 to Day 45. Groups receiving any ofthe three vORF2 vaccines and groups receiving either 4 or 8 μg of rORF2had the lowest incidence of nasal shedding of PCV2 (≦9.1%). Thechallenge control group (Group 8) had the highest shedding rate (80%),followed by the strict negative control group (Group 9), which had anincidence rate of 63.6%.

TABLE 11 Summary of Group Incidence of Nasal Shedding of PCV2 No. Ofpigs that shed Incidence Group Treatment N for at least one day Rate 1vORF2 - 16 μg (1 dose) 12 1 8.3% 2 vORF2 - 8 μg (1 dose) 12 1 8.3% 3vORF2 - 4 μg (1 dose) 12 1 8.3% 4 rORF2 - 16 μg (1 dose) 11 2 18.2%  5rORF2 - 8 μg (1 dose) 12 1 8.3% 6 rORF2 - 4 μg (1 dose) 11 1 9.1% 7 KV(2 doses) 12 5 41.7%  8 Challenge Controls 10 8  80% 9 Strict NegativeControls 11 7 63.6%  vORF2 = isolated viral ORF2; rORF2 = recombinantbaculovirus expressed ORF2; killed whole cell virus = PCV2 virus grownin suitable cell culture

The Summary of Group Incidence of Icterus, Group Incidence of GastricUlcers, Group Mean Lung Lesion Scores, and Group Incidence of LungLesions are shown below in Table 12. Six pigs died at the first testsite during the post-vaccination phase of the study (Group 4, N=1; Group6, N=1; Group 8, N=2; Group 9, N=2). Four out of six pigs had fibrinouslesions in one or more body cavities, one pig (Group 6) had lesionsconsistent with clostridial disease, and one pig (Group 9) had no grosslesions. None of the pigs that died during the post-vaccination phasedof the study had lesions consistent with PMWS.

Pigs that died post-challenge and pigs euthanized on Day 49 werenecropsied. At necropsy, icterus and gastric ulcers were not present inany group. With regard to mean % lung lesions, Group 9 had lowest mean %lung lesions (0%), followed by Group 1 with 0.40±0.50% and Group 5 with0.68±1.15%. Groups 2, 3, 7 and 8 had the highest mean % lung lesions(≦7.27%). Each of these four groups contained one pig with % lunglesions ≧71.5%, which skewed the results higher for these four groups.With the exception of Group 9 with 0% lung lesions noted, the remaining8 groups had ≦36% lung lesions. Almost all lung lesions noted weredescribed as red/purple and consolidated.

TABLE 12 Summary of Group Incidence of Icterus, Group Incidence ofGastric Ulcers, Group Mean % Lung Lesion Scores, and Group Incidence ofLung Lesions Noted Incidence of Gastric Mean % Lung Lung Lesions GroupTreatment Icterus Ulcers Lesions Noted 1 vORF2 - 16 μg (1 0/12 (0%) 0/120.40 ± 0.50% 10/12  dose) (0%) (83%) 2 vORF2 - 8 μg (1 dose) 0/12 (0%)0/12 7.41 ± 20.2% 10/12  (0%) (83%) 3 vORF2 - 4 μg (1 dose) 0/12 (0%)0/12 9.20 ± 20.9% 10/12  (0%) (83%) 4 rORF2 - 16 μg (1 0/11 (0%) 0/11 1.5 ± 4.74% 4/11 dose) (0%) (36%) 5 rORF2 - 8 μg (1 dose) 0/12 (0%)0/12 0.68 ± 1.15% 9/12 (0%) (75%) 6 rORF2 - 4 μg (1 dose) 0/11 (0%) 0/112.95 ± 5.12% 7/11 (0%) (64%) 7 KV (2 doses) 0/12 (0%) 0/12 7.27 ± 22.9%9/12 (0%) (75%) 8 Challenge Controls 0/10 (0%) 0/10 9.88 ± 29.2% 8/10(0%) (80%) 9 Strict Negative 0/11 (0%) 0/11 0/11 0/11 Controls (0%) (0%) (0%) vORF2 = isolated viral ORF2; rORF2 = recombinant baculovirusexpressed ORF2; KV or killed whole cell virus = PCV2 virus grown insuitable cell culture

The Summary of Group IHC Positive Incidence Results is shown in Table13. Group 1 (vORF2-16 μg) and Group 5 (rORF2-8 μg) had the lowest rateof IHC positive results (16.7%). Group 8 (Challenge Controls) and Group9 (Strict Negative Controls) had the highest rate of IHC positiveresults, 90% and 90.9%, respectively.

TABLE 13 Summary of Group IHC Positive Incidence Rate No. Of pigs thathad at least one tissue Incidence Group Treatment N positive for PCV2Rate 1 vORF2 - 16 μg (1 dose) 12 2 16.7% 2 vORF2 - 8 μg (1 dose) 12 325.0% 3 vORF2 - 4 μg (1 dose) 12 8 66.7% 4 rORF2 - 16 μg (1 dose) 11 436.3% 5 rORF2 - 8 μg (1 dose) 12 2 16.7% 6 rORF2 - 4 μg (1 dose) 11 436.4% 7 KV (2 doses) 12 5 41.7% 8 Challenge Controls 10 9 90.0% 9 StrictNegative Controls 11 10 90.9% vORF2 = isolated viral ORF2; rORF2 =recombinant baculovirus expressed ORF2; KV or killed whole cell virus =PCV2 virus grown in suitable cell culture

Post-challenge, Group 5, which received one dose of 8 μg of rORF2antigen, outperformed the other 6 vaccine groups. Group 5 had thehighest ADWG (0.94±0.22 lbs/day), the lowest incidence of abnormalbehavior (0%), the second lowest incidence of cough (8.3%), the lowestincidence of overall clinical symptoms (8.3%), the lowest mortality rate(0%), the lowest rate of nasal shedding of PCV2 (8.3%), the secondlowest rate for mean % lung lesions (0.68±1.15%) and the lowestincidence rate for positive tissues (16.7%). Groups receiving variouslevels of rORF2 antigen overall outperformed groups receiving variouslevels of vORF2 and the group receiving 2 doses of killed whole cellPCV2 vaccine performed the worst. Tables 14 and 15 contain summaries ofgroup post-challenge data.

TABLE 14 Summary of Group Post-Challenge Data - Part 1 Overall Incidenceof ADWG Abnormal Clinical Group N Treatment (lbs/day) Behavior CoughSymptoms 1 12 vORF2 - 16 μg 0.87 ± 0.29 2/12 (16.7%) 3/12 (25%) 41.7% (1dose) 2 12 vORF2 - 8 μg 0.70 ± 0.32 4/12 (33.3% 1/12 (8.3% 41.7% (1dose) 3 12 vORF2 - 4 μg 0.49 ± 0.21 8/12 (66.7%) 2/12 (16.7% 66.7% (1dose) 4 11 rORF2 - 16 μg 0.84 ± 0.30 3/11 (27.3%) 0/11 (0%) 36.4% (1dose) 5 12 rORF2 - 8 μg 0.94 ± 0.22 0/12 (0%) 1/12 (8.3%  8.3% (1 dose)6 11 rORF2 - 4 μg 0.72 ± 0.25 1/11 (9.1% 0/11 (0%)  9.1% (1 dose) 7 12KV 0.50 ± 0.15 7/12 (58.3) 0/12 (0%) 58.3% (2 doses) 8 10 Challenge 0.76± 0.19 1/10 (10%) 2/10 (20%   40% Controls 9 11 Strict Negative 1.06 ±0.17 0/11 (0%) 0/11 (0%)   0% Controls vORF2 = isolated viral ORF2;rORF2 = recombinant baculovirus expressed ORF2; KV or killed whole cellvirus = PCV2 virus grown in suitable cell culture

TABLE 15 Summary of Group Post-Challenge Data - Part 2 Mean % IncidenceRate of at Mortality Nasal Lung least one tissue IHC Group N TreatmentRate Shedding Lesions positive for PCV2 1 12 vORF2 - 16 μg 8.3%   8.3%0.40 ± 0.50% 16.7% (1 dose) 2 12 vORF2 - 8 μg 8.3%   8.3% 7.41 ± 20.2%25.0% (1 dose) 3 12 vORF2 - 4 μg 0% 8.3% 9.20 ± 20.9% 66.7% (1 dose) 411 rORF2 - 16 μg 0% 18.2%  1.50 ± 4.74% 36.3% (1 dose) 5 12 rORF2 - 8 μg0% 8.3% 0.68 ± 1.15% 16.7% (1 dose) 6 11 rORF2 - 4 μg 9.1%   9.1% 2.95 ±5.12% 36.4% (1 dose) 7 12 KV 16.7%   41.7%  7.27 ± 22.9% 41.7% (2 doses)8 10 Challenge 10%   80% 9.88 ± 29.2% 90.0% Controls 9 11 StrictNegative 0% 63.6%  0/11 (0%) 90.9% Controls vORF2 = isolated viral ORF2;rORF2 = recombinant baculovirus expressed ORF2; KV or killed whole cellvirus = PCV2 virus grown in suitable cell culture

Results of this study indicate that all further vaccine efforts shouldfocus on a rORF2 vaccine. Overall, nasal shedding of PCV2 was detectedpost-challenge and vaccination with a PCV2 vaccine resulted in areduction of shedding. Immunohistochemistry of selected lymphoid tissuesalso served as a good parameter for vaccine efficacy, whereas largedifferences in ADWG, clinical symptoms, and gross lesions were notdetected between groups. This study was complicated by the fact thatextraneous PCV2 was introduced at some point during the study, asevidenced by nasal shedding of PCV2, PCV2 seroconversion and positiveIHC tissues in Group 9, the strict negative control group.

Discussion

Seven PCV2 vaccines were evaluated in this study, which included threedifferent dose levels of vORF2 antigen administered once on Day 0, threedifferent dose levels of rORF2 antigen administered once on Day 0 andone dose level of killed whole cell PCV2 vaccine administered on Day 0and Day 14. Overall, Group 5, which received I dose of vaccinecontaining 8 μg of rORF2 antigen, had the best results. Group 5 had thehighest ADWG, the lowest incidence of abnormal behavior, the lowestincidence of abnormal respiration, the second lowest incidence of cough,the lowest incidence of overall clinical symptoms, the lowest mortalityrate, the lowest rate of nasal shedding of PCV2, the second lowest ratefor mean % lung lesions and the lowest incidence rate for positive IHCtissues.

Interestingly, Group 4, which received a higher dose of rORF2 antigenthan Group 5, did not perform as well or better than Group 5. Group 4had a slightly lower ADWG, a higher incidence of abnormal behavior, ahigher incidence of overall clinical symptoms, a higher rate of nasalshedding of PCV2, a higher mean % lung lesions, and a higher rate forpositive IHC tissues than Group 5. Statistical analysis, which may haveindicated that the differences between these two groups were notstatistically significant, was not conducted on these data, but therewas an observed trend that Group 4 did not perform as well as Group 5.

Post-vaccination, 6 pigs died at the first study site. Four of the sixpigs were from Group 8 or Group 9, which received no vaccine. None ofthe six pigs demonstrated lesions consistent with PMWS, no adverseevents were reported and overall, all seven vaccines appeared to be safewhen administered to pigs approximately 11 days of age. During thepost-vaccination phase of the study, pigs receiving either of three doselevels of vORF2 vaccine or killed whole cell vaccine had the highestIFAT levels, while Group 5 had the lowest IFAT levels just prior tochallenge, of the vaccine groups.

Although not formally proven, the predominant route of transmission ofPCV2 to young swine shortly after weaning is believed to be by oronasaldirect contact and an efficacious vaccine that reduces nasal shedding ofPCV2 in a production setting would help control the spread of infection.Groups receiving one of three vORF2 antigen levels and the groupreceiving 8 μg of rORF2 had the lowest incidence rate of nasal sheddingof PCV2 (8.3%). Expectedly, the challenge control group had the highestincidence rate of nasal shedding (80%).

Gross lesions in pigs with PMWS secondary to PCV2 infection typicallyconsist of generalized lymphadenopathy in combination with one or amultiple of the following: (1) interstitial pneumonia with interlobularedema, (2) cutaneous pallor or icterus, (3) mottled atrophic livers, (4)gastric ulcers, (5) nephritis and (6) reproductive disorders, e.g.abortion, stillbirths, mummies, etc. At necropsy, icterus, hepatitis,nephritis, and gastric ulcers were not noted in any groups andlymphadenopathy was not specifically examined for. The mean % lunglesion scores varied between groups. The group receiving 16 μg of vORF2antigen had the lowest mean % lung lesion score (0.40±0.50%), followedby the group that received 8 μg of rORF2 (0.68±1.15%). As expected, thechallenge control group had the highest mean % lung lesion score(9.88±29.2%). In all four groups, the mean % lung lesion scores wereelevated due to one pig in each of these groups that had very high lunglesion scores. Most of the lung lesions were described as red/purple andconsolidated. Typically, lung lesions associated with PMWS are describedas tan and non-collapsible with interlobular edema. The lung lesionsnoted in this study were either not associated with PCV2 infection or asecond pulmonary infectious agent may have been present. Within thecontext of this study, the % lung lesion scores probably do not reflecta true measure of the amount of lung infection due to PCV2.

Other researchers have demonstrated a direct correlation between thepresence of PCV2 antigen by IHC and histopathology. Histopathology onselect tissues was not conducted with this study. Group 1 (16 μg ofvORF2) and Group 5 (8 μg of rORF2) had the lowest incidence rate of pigspositive for PCV2 antigen (8.3%), while Group 9 (the strict negativecontrol group—90.9%) and Group 8 (the challenge control group—90.0%) hadthe highest incidence rates for pigs positive for PCV2 antigen. Due tothe non-subjective nature of this test, IHC results are probably one ofthe best parameters to judge vaccine efficacy on.

Thus, in one aspect of the present invention, the Minimum ProtectiveDosage (MPD) of a 1 ml/1 dose recombinant product with extracted PCV2ORF2 (rORF2) antigen in the CDCD pig model in the face of a PCV2challenge was determined. Of the three groups that received varyinglevels of rORF2 antigen, Group 5 (8 μg of rORF2 antigen) clearly had thehighest level of protection. Group 5 either had the best results or wastied for the most favorable results with regard to all of the parametersexamined. When Group 5 was compared with the other six vaccine groupspost-challenge, Group 5 had the highest ADWG (0.94±0.22 lbs/day), thelowest incidence of abnormal behavior (0%), the second lowest incidenceof cough (8.3%), the lowest incidence of overall clinical symptoms(8.3%), the lowest mortality rate (0%), the lowest rate of nasalshedding of PCV2 (8.3%), the second lowest rate for mean % lung lesions(0.68±1.15%) and the lowest incidence rate for positive IHC tissues(16.7%).

In another aspect of the present invention, the MPD of a 1 ml/l doseconventional product that is partially purified PCV2 ORF2 (vORF2)antigen in the CDCD pig model in the face of a PCV2 challenge wasdetermined. Of the three groups that received varying levels of vORF2antigen, Group 1 (16 μg of vORF2) had the highest level of protection.Group 1 outperformed Groups 2 and 3 with respect to ADWG, mean % lunglesions, and IHC. Groups 1 and 2 (8 μg of vORF2 antigen) performedequally with respect to overall incidence of clinical symptoms, Group 3(4 μg of vORF2 antigen) had the lowest mortality rate, and all threegroups performed equally with respect to nasal shedding. Overall, vORFvaccines did not perform as well as rORF vaccines.

In yet another aspect of the present invention, the efficacy of amaximum dose of a 2 ml/2 dose Conventional Killed PCV2 vaccine in theCDCD pig model in the face of a PCV2 challenge was determined. Of theseven vaccines evaluated in this study, the killed whole cell PCV2vaccine performed the worst. Piglets receiving two doses of killed wholecell PCV2 vaccine had the lowest ADWG, the second highest rate ofabnormal behavior (58.3%), the second highest overall incidence ofclinical symptoms (58.3%), the highest mortality rate (16.7%), thesecond highest incidence of nasal shedding (41.7%), highest mean % lunglesions (9.88±29.2%), a high incidence of lung lesions noted (75%) and amoderate IHC incidence rate in tissues (41.7%). However, it was stilleffective at invoking an immune response.

In still another aspect of the present invention, nasal shedding of PCV2was assessed as an efficacy parameter and the previous PCV2 efficacyparameters from previous studies were reconfirmed. Results from thisstudy indicate that nasal shedding of PCV2 occurs following intra nasalchallenge and that PCV2 vaccines reduce nasal shedding of PCV2post-challenge. Furthermore, results from this study and reports in theliterature indicate that IHC should continue to be evaluated in futurePCV2 vaccine trials as well.

Some additional conclusions arising from this study are thatlymphadenopathy is one of the hallmarks of PMWS. Another one of thehallmarks of PMWS is lymphoid depletion and multinucleated/gianthistiocytes. Additionally, no adverse events or injection site reactionswere noted for any of the 7 PCV2 vaccines and all 7 PCV2 vaccinesappeared to be safe when administered to young pigs.

Example 5

This example tests the efficacy of eight PCV2 candidate vaccines andreconfirms PCV2 challenge parameters from earlier challenge studiesfollowing exposure to a virulent strain of PCV2. One hundred and fully(150) cesarean derived colostrum deprived (CDCD) piglets, 6-16 days ofage, were blocked by weight and randomly divided into 10 groups of equalsize. Table 16 sets forth the General Study Design for this Example.

TABLE 16 General Study Design Challenge with KLH/ICFA Virulent PRRSVNecropsy No. Of Day of on Day 22 PCV2 on MLV on on Day Group PigsTreatment Treatment and Day 28 Day 25 Day 46 50 1 15 PVC2 Vaccine 1 0 &14 + + + + 16 μg rORF2 - IMS 1314 2 15 PVC2 Vaccine 2 0 & 14 + + + + 16μg vORF2 - Carbopol 3 15 PCV2 Vaccine 3 0 & 14 + + + + 16 μg rORF2 -Carbopol 4 15 PCV2 Vaccine 2 0 + + + + 16 μg vORF2 - Carbopol 5 15 PVC2Vaccine 3 0 & 14 + + + + 4 μg rORF2 - Carbopol 6 15 PVC2 Vaccine 3 0 &14 + + + + 1 μg rORF2 - Carbopol 7 15 PVC2 Vaccine 3 0 & 14 + + + + 0.25μg rORF2 - Carbopol 8 15 PVC2 Vaccine 4 > 0 & 14 + + + + 8.0 log KV -Carbopol 9 15 Challenge N/A + + + + Controls 10 15 None - Strict N/A +− + + Negative Control Group vORF2 = isolated viral ORF2; rORF2 =recombinant baculovirus expressed ORF2; KV or killed whole cell virus =PCV2 virus grown in suitable cell culture

The vaccine formulations given to each group were as follows. PCV2Vaccine No. 1, administered at 1×2 ml dose to Group 1, was a high dose(16 ug/2 ml dose) of inactivated recombinant ORF2 antigen adjuvantedwith IMS 1314 (16 ug rORF2—IMS 1314). PCV2 Vaccine No. 2, administeredat 1×2 ml dose to Group 2, was a high dose (16 ug (2 ml dose) of apartially purified VIDO R-1 generated PCV2 ORF2 antigen adjuvanted withCarbopol (16 ug vORF2—Carbopol). PCV2 Vaccine No. 3, administered at 1×2ml dose to Group 3, was a high dose (16 ug/2 ml dose) of inactivatedrecombinant ORF2 antigen adjuvanted with Carbopol (16 ugrORF2—Carbopol). PCV2 Vaccine No. 4, administered at 1×1 ml dose toGroup 4, was a high dose (16 ug/1 ml dose) of a partially purified VIDOR-1 generated PCV2 ORF2 antigen adjuvanted with Carbopol (16 ugvORF2—Carbopol). Vaccine No. 5, administered at 1×2 ml dose to Group 5,was a 4 ug/2 ml dose of an inactivated recombinant ORF2 antigenadjuvanted with Carbopol (4 ug rORF2—Carbopol). PCV2 Vaccine No. 6,administered at 1×2 ml dose to Group 6, was a 1 ug/2 ml dose of aninactivated recombinant ORF2 antigen adjuvanted with Carbopol (1 ugrORF2—Carbopol). PCV2 Vaccine No. 7, administered at 1×2 ml dose toGroup 7, was a low dose (0.25 ug/2 ml dose) of inactivated recombinantORF2 antigen adjuvanted with Carbopol (0.25 ug rORF2—Carbopol). PCV2Vaccine No. 8, administered at 1×2 ml dose to Group 8, was a high dose(pre-inactivation titer>8.0 log/2 ml dose) Inactivated ConventionalKilled VIDO R-1 generated PCV2 Struve antigen adjuvanted with Carbopol(>8.0 log KV—Carbopol). On Day 0, Groups 1-8 were treated with theirassigned vaccines. Groups 1-3 and 5-8 received boosters of theirrespective vaccines again on Day 14. The effectiveness of a single doseof 16 μg of vORF2—Carbopol was tested on Group 4 which did not receive abooster on Day 14. Piglets were observed for adverse events andinjection site reactions following both vaccinations. On Day 21 thepiglets were moved to a second study site where Groups 1-9 were grouphoused in one building and Group 10 was housed in a separate building.All pigs received keyhole limpet hemocyanin emulsified with incompleteFreund's adjuvant (KLH/ICFA) on Days 22 and 28. On Day 25, Groups 1-9were challenged with approximately 4 logs of virulent PCV2 virus. By Day46, very few deaths had occurred in the challenge control group. In anattempt to immunostimulate the pigs and increase the virulence of thePCV2 challenge material, all Groups were treated with INGELVAC® PRRSVMLV (Porcine Reproductive and Respiratory Vaccine, Modified Live Virus)on Day 46.

Pre- and post-challenge blood samples were collected for PCV2 serology.Post-challenge, body weight data for determination of average dailyweight gain (ADWG) and observations of clinical signs were collected. OnDay 50, all surviving pigs were necropsied, gross lesions were recorded,lungs were scored for pathology, and selected tissues were preserved informalin for examination by Immunohistochemistry (IHC) for detection ofPCV2 antigen at a later date.

Materials and Methods

This was a partially-blind vaccination-challenge feasibility studyconducted in CDCD pigs, 6 to 1.6 days of age on Day 0. To be included inthe study, PCV2 IFA titers of sows were ≦1:1000. Additionally, theserologic status of sows were from a known PRRS-negative herd. Sixteen(16) sows were tested for PCV2 serological status and all sixteen (16)had a PCV2 titer of ≦1000 and were transferred to the first study site.One hundred fifty (150) piglets were delivered by cesarean sectionsurgeries and were available for this study on Day 3. On Day −3, 150CDCD pigs at the first study site were weighed, identified with eartags, blocked by weight and randomly assigned to 1 of 10 groups, as setforth above in table 16. Blood samples were collected from all pigs. Ifany test animal meeting the inclusion criteria was enrolled in the studyand was later excluded for any reason, the Investigator and Monitorconsulted in order to determine the use of data collected from theanimal in the final analysis. The date of which enrolled piglets wereexcluded and the reason for exclusion was documented. No sows meetingthe inclusion criteria, selected for the study and transported to thefirst study site were excluded. No piglets were excluded from the study,and no test animals were removed from the study prior to termination.Table 17 describes the time frames for the key activities of thisExample.

TABLE 17 Study Activities Study Day Actual Dates Study Activity −3 Apr.04, 2003 Weighed pigs; health exam; randomized to groups; collectedblood samples −3, Apr. 04, 2003 Observed for overall health and foradverse events post-  0-21 Apr. 07, 2003 to vaccination May 27, 2003  0Apr. 07, 2003 Administered respective IVPs to Groups 1-8 0-7 Apr. 07,2003 to Observed pigs for injection site reactions Apr. 14, 2003 14 Apr.21, 2003 Boostered Groups 1-3, 5-8 with respective IVPs; blood sampledall pigs 14-21 Apr. 21, 2003 to Observed pigs for injection reactionsApr. 28, 2003 19-21 Apr. 26, 2003 to Treated all pigs with antibioticsApr. 28, 2003 21 Apr. 28, 2003 Pigs transported from Struve Labs, Inc.to Veterinary Resources, Inc.(VRI) 22-50 Apr. 28, 2003 to Observed pigsfor clinical signs post-challenge May 27, 2003 22 Apr. 29, 2003 TreatedGroups 1-10 with KLH/ICFA 25 May 02, 2003 Collected blood samples fromall pigs; weighed all pigs; challenged Groups 1-9 with PCV2 challengematerial 28 May 05, 2003 Treated Groups 1-10 with KLH/ICFA 32 May 09,2003 Collected blood samples from all pigs 46 May 23, 2003 AdministeredINGELVAC ® PRRS MLV to all groups 50 May 27, 2003 Collected bloodsamples, weighed and necropsied all pigs; gross lesions were recorded;lungs were evaluated for lesions; fresh and formalin fixed tissuesamples were saved; In-life phase of the study was completed

Following completion of the in-life phase of the study, formalin fixedtissues were examined by Immunohistochemistry (IHC) for detection ofPCV2 antigen by a pathologist, blood samples were evaluated for PCV2serology, and average daily weight gain (ADWG) was determined from Day25 to Day 50.

Animals were housed at the first study site in individual cages in sevenrooms from birth to approximately 11 days of age (approximately Day 0 ofthe study). Each room was identical in layout and consisted of stackedindividual stainless steel cages with heated and filtered air suppliedseparately to each isolation unit. Each room had separate heat andventilation, thereby preventing cross-contamination of air betweenrooms. Animals were housed in two different buildings at the secondstudy site. Group 10 (The Strict negative control group) was housedseparately in a converted nursery building and Groups 1-9 were housed ina converted farrowing building. Each group was housed in a separate pen(14-15 pigs per pen) and each pen provided approximately 2.3 square feetper pig. Groups 2, 4 and 8 were penned in three adjacent pens on oneside of the alleyway and Groups 1, 3, 5, 6, 7, and 9 were penned in sixadjacent pens on the other side of the alleyway. The Group separationwas due to concern by the Study Monitor that vaccines administered toGroups 2, 4, and 8 had not been fully inactivated. Each pen was on anelevated deck with plastic slatted floors. A pit below the pens servedas a holding tank for excrement and waste. Each building had its ownseparate heating and ventilation systems, with little likelihood ofcross-contamination of air between buildings.

At the first study site, piglets were fed a specially formulated milkration from birth to approximately 3 weeks of age. All piglets wereconsuming solid, special mixed ration by Day 21 (approximately 4½ weeksof age). At the second study site, all piglets were fed a customnon-medicated commercial mix ration appropriate for their age andweight, ad libitum. Water at both study sites was also available adlibitum.

All test pigs were treated with 1.0 mL of NAXCEL®, IM, in alternatinghams on Days 19, 20, and 21. In addition, Pig No. 11 (Group 1) wastreated with 0.5 mL of NAXCEL® IM on Day 10, Pig No. 13 (Group 1.0) wastreated with 1 mL of Penicillin and 1 mL of PREDEF® 2× on Day 10, PigNo. 4 (Group 9) was treated with 1.0 mL of NAXCEL® IM on Day 11, andPigs 1 (Group 1), 4 and 11 were each treated with 1.0 mL of NAXCEL® onDay 14 for various health reasons.

While at both study sites, pigs were under veterinary care. Animalhealth examinations were conducted on Day −3 and were recorded on theHealth Examination Record Form. All animals were in good health andnutritional status before vaccination as determined by observation onDay 0. All test animals were observed to be in good health andnutritional status prior to challenge. Carcasses and tissues weredisposed of by rendering. Final disposition of study animals wasrecorded on the Animal Disposition Record.

On Days 0 and 14, pigs assigned to Groups 1-3 and 5-8 received 2.0 mL ofassigned PCV2 Vaccines 14, respectively, IM in the right and left neckregion, respectively, using a sterile 3.0 mL Luer-lock syringe and asterile 20 g×2½″ needle. Pigs assigned to Group 4 received 1.0 mL ofPCV2 Vaccine No. 2, IM in the right neck region using a sterile 3.0 mLLuer-lock syringe and a sterile 20 g×½″ needle on Day 0 only.

On Day 22 all test pigs received 2.0 mL of KLH/ICFA IM in the left neckregion using a sterile 3.0 mL Luer-lock syringe and a sterile 20 g×1″needle. On Day 28 all test pigs received 2.0 mL of KLH/ICFA in the rightham region using a sterile 3.0 mL Luer-lock syringe and a sterile 20g×1″ needle.

On Day 25, pigs assigned to Groups 1-9 received 1.0 mL of PCV2 ISUVDLchallenge material (3.98 log₁₀ TCID₅₀/mL) IM in the right neck regionusing a sterile 3.0 mL Luer-lock syringe and a sterile 20 g×1″ needle.An additional 1.0 mL of the same material was administered IN to eachpig (0.5 mL per nostril) using a sterile 3.0 mL Luer-lock syringe andnasal canula.

On Day 46, all test pigs received 2.0 mL INGELVAC® PRRS MLV, IM, in theright neck region using a sterile 3.0 mL Luer0lock syringe and a sterile20 g×1″ needle. The PRRSV MLV was administered in an attempt to increasevirulence of the PCV2 challenge material.

Test pigs were observed daily for overall health and adverse events onDay −3 and from Day 0 to Day 21. Each of the pigs were scored for normalor abnormal behavior, respiration, or cough. Observations were recordedon the Clinical Observation Record. All test pigs were observed from Day0 to Day 7, and Group 7 was further observed from Day 14 to 21, forinjection site reactions. Average daily weight gain was determined byweighing each pig on a calibrated scale on Days −3, 25 and 50, or on theday that a pig was found dead after challenge. Body weights wererecorded on the Body Weight Form. Day −3 body weights were utilized toblock pigs prior to randomization. Day 25 and Day 50 weight data wasutilized to determine the average daily weight gain (ADWG) for each pigduring these time points. For pigs that died after challenge and beforeDay 50, the ADWG was adjusted to represent the ADWG from Day 25 to theday of death.

In order to determine PCV2 serology, venous whole blood was collectedfrom each piglet from the orbital venous sinus on Days −3 and 14. Foreach piglet, blood was collected from the orbital venous sinus byinserting a sterile capillary tube into the medial canthus of one of theeyes and draining approximately 3.0 mL of whole blood into a 4.0 mLSerum Separator Tube (SST). On Days 25, 32, and 50, venous whole bloodfrom each pig was collected from the anterior vena cava using a sterile20 g×1½″ Vacutainer® needle (Becton Dickinson and Company, FranklinLakes, N.J.), a Vaccutainer® needle holder and a 13 mL SST. Bloodcollections at each time point were recorded on the Sample CollectionRecord. Blood in each SST was allowed to clot, each SST was then spundown and the serum harvested. Harvested serum was transferred to asterile snap tube and stored at −70±10° C. until tested at a later date.Serum samples were tested for the presence of PCV2 antibodies byBIVI-R&D personnel.

Pigs were observed once daily from Day 22 to Day 50 for clinicalsymptoms and scored for normal or abnormal behavior, respiration orcough. Clinical observations were recorded on the Clinical ObservationRecord.

Pigs Nos. 46 (Group 1) and 98 (Groups 9) died at the first study site.Both of these deaths were categorized as bleeding deaths and necropsieswere not conducted on these two pigs. At the second study site, pigsthat died after challenge and prior to Day 50, and pigs euthanized onDay 50, were necropsied. Any gross lesions were noted and thepercentages of lung lobes with lesions were recorded on the NecropsyReport Form.

From each of the pigs necropsied at the second study site, a tissuesample of tonsil, lung, heart, and mesenteric lymph node was placed intoa single container with buffered 10% formalin; while another tissuesample from the same aforementioned organs was placed into a Whirl-pak®(M-Tech Diagnostics Ltd., Thelwall, UK) and each Whirl-pak® was placedon ice. Each container was properly labeled. Sample collections wererecorded on the Necropsy Report Form. Afterwards, formalin-fixed tissuesamples and a Diagnostic Request Form were submitted for IHC testing.IHC testing was conducted in accordance with standard laboratoryprocedures for receiving samples, sample and slide preparation, andstaining techniques. Fresh tissues in Whirl-paks® were shipped with icepacks to the Study Monitor for storage (−70°±10° C.) and possible futureuse.

Formalin-fixed tissues were examined by a pathologist for detection ofPCV2 by IHC and scored using the following scoring system: 0=None;1=Scant positive staining, few sites; 2=Moderate positive staining,multiple sites; and 3=Abundant positive staining, diffuse throughout thetissue. For analytical purposes, a score of 0 was considered “negative,”and a score of greater than 0 was considered “positive.”

Results

Results for this example are given below. It is noted that Pigs No. 46and 98 died on days 14 and 25 respectively. These deaths werecategorized as bleeding deaths. Pig No. 11 (Group 1) was panting withrapid respiration on Day 15. Otherwise, all pigs were normal forbehavior, respiration and cough during this observation period and nosystemic adverse events were noted with any groups. No injection sitereactions were noted following vaccination on Day 0. Followingvaccination on Day 14, seven (7) out of fourteen (14) Group 1 pigs(50.0%) had swelling with a score of “2” on Day 15. Four (4) out offourteen (14) Group 1 (28.6%) still had a swelling of “2” on Day 16.None of the other groups experienced injection site reactions followingeither vaccination.

Average daily weight gain (ADWG) results are presented below in Table18. Pig Nos. 46 and 98 that died from bleeding were excluded from groupresults. Group 4, which received one dose of 16 ug vORF2—Carbopol, hadthe highest ADWG (1.16±0.26 lbs/day), followed by Groups 1, 2, 3, 5, 6,and 10 which had ADWGs that ranged from 1.07±0.23 lbs/day to 1.11±10.26lbs/day. Group 9 had the lowest ADWG (0.88±0.29 lbs/day), followed byGroups 8 and 7, which had ADWGs of 0.93±0.33 lbs/day and 0.99±0.44lbs/day, respectively.

TABLE 18 Summary of Group Average Daily Weight Gains (ADWG) ADWG -lbs/day (Day 25 to Day 50) or adjusted for pigs Group Treatment N deadbefore Day 50 1 rORF2 - 16 μg - IMS 1314 2 doses 14 1.08 ± 0.30 lbs/day2 vORF2 - 16 μg - Carbopol 2 doses 15 1.11 ± 0.16 lbs/day 3 rORF2 - 16μg - Carbopol 2 doses 15 1.07 ± 0.21 lbs/day 4 vORF2 - 16 μg - Carbopol1 dose 15 1.16 ± 0.26 lbs/day 5 rORF2 - 4 μg - Carbopol 1 dose 15 1.07 ±0.26 lbs/day 6 rORF2 - 1 μg - Carbopol 2 doses 15 1.11 ± 0.26 lbs/day 7rORF2 - 0.25 μg - Carbopol 2 doses 15 0.99 ± 0.44 lbs/day 8 KV > 8.0log - Carbopol 2 doses 15 0.93 ± 0.33 lbs/day 9 Challenge Controls 140.88 ± 0.29 lbs/day 10 Strict Negative Controls 15 1.07 ± 0.23 lbs/dayvORF2 = isolated viral ORF2; rORF2 = recombinant baculovirus expressedORF2; KV or killed whole cell virus = PCV2 virus grown in suitable cellculture

PVC2 serology results are presented below in Table 19. All ten (10)groups were seronegative for PCV2 on Day −3. On Day 14, PCV2 titersremained low for all ten (10) groups (range of 50-113). On Day 25, Group8, which received the whole cell killed virus vaccine, had the highestPCV2 titer (4617), followed by Group 2, which received 16 ugvORF2—Carbopol, Group 4, which received as single dose of 16 ugvORF2—Carbopol, and Group 3, which received 16 ug rORF2—Carbopol, whichhad titers of 2507, 1920 and 1503 respectively. On Day 32 (one week postchallenge), titers for Groups 1-6 and Group 8 ranged from 2360 to 7619;while Groups 7 (0.25 ug rORF2—Carbopol), 9 (Challenge Control), and 10(Strict negative control) had titers of 382, 129 and 78 respectively. OnDay 50 (day of necropsy), all ten (10) groups demonstrated high PCV2titers (>1257).

On Days 25, 32, and 50, Group 3, which received two doses of 16 ugrORF2—Carbopol, had higher antibody titers than Group 1, which receivedtwo doses of 16 ug rORF2—IMS 1314. On Days 25, 32 and 50, Group 2, whichreceived two doses of 16 ug vORF2, had higher titers than Group 4, whichreceived only one does of the same vaccine. Groups 3, 5, 6, 7, whichreceived decreasing levels of rORF2—Carbopol, of 16, 4, 1, and 0.25 ugrespectively, demonstrated correspondingly decreasing antibody titers onDays 25 and 32.

TABLE 19 Summary of Group PCV2 IFA Titers Day Group Treatment Day −3 Day14** Day 25*** Day 32 50**** 1 rORF2 - 16 μg - 50 64 646 3326 4314 IMS1314 2 doses 2 vORF2 - 16 μg - 50 110 2507 5627 4005 Carbopol 2 doses 3rORF2 - 16 μg - 50 80 1503 5120 6720 Carbopol 2 doses 4 vORF2 - 16 μg -50 113 1920 3720 1257 Carbopol 1 dose 5 rORF2 - 4 μg - 50 61 1867 39334533 Carbopol 1 dose 6 rORF2 - 1 μg - 50 70 490 2360 5740 Carbopol 2doses 7 rORF2 - 0.25 μg - 50 73 63 382 5819 Carbopol 2 doses 8 KV > 8.0log - Carbopol 50 97 4617 7619 10817 2 doses 9 Challenge Controls 50 5350 129 4288 10 Strict Negative Controls 50 50 50 78 11205 vORF2 =isolated viral ORF2; rORF2 = recombinant baculovirus expressed ORF2; KVor killed whole cell virus = PCV2 virus grown in suitable cell culture*For calculation purposes, a ≦ 100 IFA titer was designated as a titerof “50”; a ≧ 6400 IFA titer was designated as a titer of “12,800”. **Dayof Challenge ***Day of Necropsy

The results from the post-challenge clinical observations are presentedbelow. Table 20 includes observations for Abnormal Behavior, AbnormalRespiration, Cough and Diarrhea Table 21 includes the results from theSummary of Group Overall Incidence of Clinical Symptoms and Table 22includes results from the Summary of Group Mortality RatesPost-challenge. The incidence of abnormal behavior, respiration andcough post-challenge were low in pigs receiving 16 ug rORF2-IMS 1314(Group 1), 16 ug rORF2-Carbopol (Group 3), 1 ug rORF2-Carbopol (Group6), 0.25 ug rORF2-Carbopol (Group 7), and in pigs in the ChallengeControl Group (Group 9). The incidence of abnormal behavior,respiration, and cough post-challenge was zero in pigs receiving 16 ugvORF2-Carbopol (Group 2), a single dose of 16 ug vORF2-Carbopol (Group4), 4 ug rORF2-Carbopol (Group 5), >8 log KV-Carbopol (Group 8), and inpigs in the strict negative control group (Group 10).

The overall incidence of clinical symptoms varied between groups. Pigsreceiving 16 ug vORF2-Carbopol (Group 2), a single dose of 16 ugvORF2-Carbopol (Group 4), and pigs in the Strict negative control group(Group 10) had incidence rates of 0%; pigs receiving 16 ugrORF2-Carbopol (Group 3), and 1 ug rORF2-Carbopol (Group 6) hadincidence rates of 6.7%; pigs receiving 16 ug rORF2-IMS 1314 (Group 1)had an overall incidence rate of 7.1%; pigs receiving 4 ugrORF2-Carbopol (Group 5), 0.25 ug rORF2-Carbopol (Group 7), and >8 logKV vaccine had incidence rates of 13.3%; and pigs in the ChallengeControl Group (Group 9) had an incidence rate of 14.3%.

Overall mortality rates between groups varied as well. Group 8, whichreceived 2 doses of KV vaccine had the highest mortality rate of 20.0%;followed by Group 9, the challenge control group, and Group 7, whichreceived 0.25 ug rORF2-Carbopol and had mortality rates of 14.3% and13.3% respectively. Group 4, which received one dose of 16 ugvORF2—Carbopol had a 6.7% mortality rate. All of the other Groups, 1, 2,3, 5, 6, and 10, had a 0% mortality rate.

TABLE 20 Summary of Group Observations for Abnormal Behavior, AbnormalRespiration, and Cough Post-Challenge Abnormal Abnormal Group TreatmentN Behavior¹ Behavior² Cough³ 1 rORF2 - 16 μg - 14 0/14 0/14 1/14 IMS1314 2 doses (0%) (0%) (7.1%)   2 vORF2 - 16 μg - 15 0/15 0/15 0/15Carbopol 2 doses (0%) (0%) (0%) 3 rORF2 - 16 μg - 15 0/15 0/15 1/15Carbopol 2 doses (0%) (0%) (6.7%)   4 vORF2 - 16 μg - 15 0/15 0/15 0/15Carbopol 1 dose (0%) (0%) (0%) 5 rORF2 - 4 μg - 15 1/15 1/15 0/15Carbopol 1 dose (6.7%)   (6.7%)   (0%) 6 rORF2 - 1 μg - 15 0/15 0/151/15 Carbopol 2 doses (0%) (0%) (6.7%)   7 rORF2 - 0.25 μg - 15 0/151/15 1/15 Carbopol 2 doses (0%) (6.7%)   (06.7%)   8 KV > 8.0 log - 151/15 1/15 0/15 Carbopol 2 doses (6.7%)   (6.7%)   (0%) 9 ChallengeControls 14 1/14 1/14 2/14 (7.1%)   (7.1%)   (14/3%)  10 Strict Negative15 0/15 0/15 0/15 Controls (0%) (0%) (0%) ¹Total number of pigs in eachgroup that demonstrated any abnormal behavior for at least one day²Total number of pigs in each group that demonstrated any abnormalrespiration for at least one day ³Total number of pigs in each groupthat demonstrated a cough for at least one day

TABLE 21 Summary of Group Overall Incidence of Clinical Symptoms Post-Challenge Incidence of pigs with Clinical Incidence Group Treatment NSymptoms¹ Rate 1 rORF2 - 16 μg - 14 1 7.1% IMS 1314 2 doses 2 vORF2 - 16μg - Carbopol 2 15 0 0.0% doses 3 rORF2 - 16 μg - Carbopol 2 15 1 6.7%doses 4 vORF2 - 16 μg - Carbopol 1 15 0 0.0% dose 5 rORF2 - 4 μg - 15 213.3% Carbopol 1 dose 6 rORF2 - 1 μg - 15 1 6.7% Carbopol 2 doses 7rORF2 - 0.25 μg - Carbopol 15 2 13.3% 2 doses 8 KV > 8.0 log - Carbopol2 15 2 13.3% doses 9 Challenge Controls 14 2 14.3% 10 Strict NegativeControls 15 0 0.0% vORF2 = isolated viral ORF2; rORF2 = recombinantbaculovirus expressed ORF2; KV or killed whole cell virus = PCV2 virusgrown in suitable cell culture ¹Total number of pigs in each group thatdemonstrated any clinical symptom for at least one day

TABLE 22 Summary of Group Mortality Rates Post-Challenge Dead Post-Mortality Group Treatment N challenge Rate 1 rORF2 - 16 μg - 14 0 0.0%IMS 1314 2 doses 2 vORF2 - 16 μg - Carbopol 2 15 0 0.0% doses 3 rORF2 -16 μg - Carbopol 2 15 0 0.0% doses 4 vORF2 - 16 μg - Carbopol 1 15 16.7% dose 5 rORF2 - 4 μg - 15 0 0.0% Carbopol 1 dose 6 rORF2 - 1 μg - 150 0.0% Carbopol 2 doses 7 rORF2 - 0.25 μg - Carbopol 2 15 2 13.3% doses8 KV > 8.0 log - Carbopol 2 15 3 20.0% doses 9 Challenge Controls 14 214.3% 10 Strict Negative Controls 1.5 0 0.0% vORF2 = isolated viralORF2; rORF2 = recombinant baculovirus expressed ORF2; KV or killed wholecell virus = PCV2 virus grown in suitable cell culture

The Summary of Group Mean Percentage Lung Lesions and TentativeDiagnosis is given below in Table 23. Group 9, the challenge controlgroup, had the highest percentage lung lesions with a mean of10.81±23.27%, followed by Group 7, which received 0.25 ug rORF2—Carbopoland had a mean of 6.57±24.74%, Group 5, which received 4 ugrORF2-Carbopol and had a mean of 2.88±8.88%, and Group 8, which receivedthe KV vaccine and had a mean of 2.01±4.98%. The remaining six (6)groups had lower mean percentage lung lesions that ranged from0.11±0.38% to 0.90±0.15%.

Tentative diagnosis of pneumonia varied among the groups. Group 3, whichreceived two doses of 16 ug rORF2-Carbopol, had the lowest tentativediagnosis of pneumonia, with 13.3%. Group 9, the challenge controlgroup, had 50% of the group tentatively diagnosed with pneumonia,followed by Group 10, the strict negative control group and Group 2,which received two doses of 16 ug vORF2-Carbopol, with 46.7% and 40%respectively, tentatively diagnosed with pneumonia.

Groups 1, 2, 3, 5, 9, and 10 had 0% of the group tentatively diagnosedas PCV2 infected; while Group 8, which received two doses if KV vaccine,had the highest group rate of tentative diagnosis of PCV2 infection,with 20%. Group 7, which received two doses of 0.25 ug rORF2-Carbopol,and Group 4, which received one dose of 16 ug vORF2-Carbopol hadtentative group diagnoses of PCV2 infection in 13.3% and 6.7% of eachgroup, respectively.

Gastric ulcers were only diagnosed in one pig in Group 7 (6.7%); whilethe other 9 groups remained free of gastric ulcers.

TABLE 23 Summary of Group Mean % Lung Lesion and Tentative Diagnosis No.Of pigs that shed Incidence Group Treatment N for at least one day Rate1 rORF2 - 16 μg - 15 0   0% IMS 1314 2 doses 2 vORF2 - 16 μg - 15 1 6.7% Carbopol 2 doses 3 rORF2 - 16 μg - 15 3 20.0% Carbopol 2 doses 4vORF2 - 16 μg - 15 2 13.3% Carbopol 1 dose 5 rORF2 - 4 μg - 15 3 20.0%Carbopol 1 dose 6 rORF2 - 1 μg - 15 6 40.0% Carbopol 2 doses 7 rORF2 -0.25 μg - 15 7 46.7% Carbopol 2 doses 8 KV > 8.0 log - Carbopol 15 12  80% 2 doses 9 Challenge Controls 14 14 100.0%  10 Strict NegativeControls 15 14 93.3% vORF2 = isolated viral ORF2; rORF2 = recombinantbaculovirus expressed ORF2; KV or killed whole cell virus = PCV2 virusgrown in suitable cell culture

The Summary of Group IHC Positive Incidence Results is shown below inTable 24. Group 1 (16 ug rORF2—IMS 1314) had the lowest group rate ofIHC positive results with 0% of the pigs positive for PCV2, followed byGroup 2 (16 ug vORF2—Carbopol) and Group 4 (single dose 16 ugvORF2—Carbopol), which had group IHC rates of 6.7% and 13.3%respectively. Group 9, the challenge control group, had the highest IHCpositive incidence rate with 100% of the pigs positive for PCV2,followed by Group 10, the strict negative control group, and Group 8 (KVvaccine), with 93.3% and 80% of the pigs positive for PCV2,respectively.

TABLE 24 Summary of Group IHC Positive Incidence Rate No. Of pigs thatshed Incidence Group Treatment N for at least one day Rate 1 rORF2 - 16μg - 15 0   0% IMS 1314 2 doses 2 vORF2 - 16 μg - 15 1  6.7% Carbopol 2doses 3 rORF2 - 16 μg - 15 3 20.0% Carbopol 2 doses 4 vORF2 - 16 μg - 152 13.3% Carbopol 1 dose 5 rORF2 - 4 μg - 15 3 20.0% Carbopol 1 dose 6rORF2 - 1 μg - 15 6 40.0% Carbopol 2 doses 7 rORF2 - 0.25 μg - 15 746.7% Carbopol 2 doses 8 KV > 8.0 log - Carbopol 15 12   80% 2 doses 9Challenge Controls 14 14 100.0%  10 Strict Negative Controls 15 14 93.3%vORF2 = isolated viral ORF2; rORF2 = recombinant baculovirus expressedORF2; KV or killed whole cell virus = PCV2 virus grown in suitable cellculture

Discussion

Seven PCV2 vaccines were evaluated in this example, which included ahigh dose (16 μg) of rORF2 antigen adjuvanted with IMS 1314 administeredtwice, a high dose (16 μg) of vORF2 antigen adjuvanted with Carbopoladministered once to one group of pigs and twice to a second group ofpigs, a high dose (16 μg) of rORF2 antigen adjuvanted with Carbopoladministered twice, a 4 μg dose of rORF2 antigen adjuvanted withCarbopol administered twice, a 1 μg dose of rORF2 antigen adjuvantedwith Carbopol administered twice, a low dose (0.25 μg) of rORF2 antigenadjuvanted with Carbopol administered twice, and a high dose (>8 log) ofkilled whole cell PCV2 vaccine adjuvanted with Carbopol. Overall, Group1, which received two doses of 16 μg rORF2—IMS 1314, performed slightlybetter than Groups 2 through 7, which received vaccines containingvarious levels of either vORF2 or rORF2 antigen adjuvanted with Carbopoland much better than Group 8, which received two doses of killed wholecell PCV2 vaccine. Group 1 had the third highest ADWG (1.80±0.30lbs/day), the lowest incidence of abnormal behavior (0%), the lowestincidence of abnormal respiration (0%), a low incidence of cough (7.1%),a low incidence of overall clinical symptoms (7.1%), was tied with threeother groups for the lowest mortality rate (0%), the second lowest ratefor mean % lung lesions (0.15±0.34%), the second lowest rate forpneumonia (21.4%) and the lowest incidence rate for positive IHC tissues(0%). Group 1 was, however, the only group in which injection sitereactions were noted, which included 50% of the vaccinates 1 day afterthe second vaccination. The other vaccines administered to Groups 2through 7 performed better than the killed vaccine and nearly as well asthe vaccine administered to Group 1.

Group 8, which received two doses of killed PCV2 vaccine adjuvanted withCarbopol, had the worst set of results for any vaccine group. Group 8had the lowest ADWG (0.93±0.33 lbs/day), the second highest rate ofabnormal behavior (6.7%), the highest rate of abnormal respiration(6.7%), was tied with three other groups for the highest overallincidence rate of clinical symptoms (13.3%), had the highest mortalityrate of all groups (20%), and had the highest positive IHC rate (80%) ofany vaccine group. There was concern that the killed whole cell PCV2vaccine may not have been fully inactivated prior to administration toGroup 8, which may explain this group's poor results. Unfortunately,definitive data was not available to confirm this concern. Overall, inthe context of this example, a Conventional Killed PCV2 vaccine did notaid in the reduction of PCV2 associated disease.

As previously mentioned, no adverse events were associated with the testvaccines with exception of the vaccine adjuvanted with IMS 1314.Injection site reactions were noted in 50.0% of the pigs 1 day after thesecond vaccination with the vaccine formulated with IMS 1314 and in28.6% of the pigs 2 days after the second vaccination. No reactions werenoted in any pigs receiving Carbopol adjuvanted vaccines. Any furtherstudies that include pigs vaccinated with IMS 1314 adjuvanted vaccinesshould continue to closely monitor pigs for injection site reactions.

All pigs were sero-negative for PCV2 on Day −3 and only Group 2 had atiter above 100 on Day 14. On Day 25 (day of challenge), Group 8 had thehighest PCV2 antibody titer (4619), followed by Group 2 (2507). With theexception of Groups 7, 9 and 10, all groups demonstrated a strongantibody response by Day 32. By Day 50, all groups including Groups 7, 9and 10 demonstrated a strong antibody response.

One of the hallmarks of late stage PCV2 infection and subsequent PMWSdevelopment is growth retardation in weaned pigs, and in severe cases,weight loss is noted. Average daily weight gain of groups is aquantitative method of demonstrating growth retardation or weight loss.In this example, there was not a large difference in ADWG betweengroups. Group 8 had the lowest ADWG of 0.88±0.29 lbs/day, while Group 4had the highest ADWG of 1.16±0.26 lb/day. Within the context of thisstudy there was not a sufficient difference between groups to basefuture vaccine efficacy on ADWG.

In addition to weight loss—dyspnea, leghargy, pallor of the skin andsometimes icterus are clinical symptoms associated with PMWS. In thisexample, abnormal behavior and abnormal respiration and cough were notedinfrequently for each group. As evidenced in this study, this challengemodel and challenge strain do not result in overwhelming clinicalsymptoms and this is not a strong parameter on which to base vaccineefficacy.

Overall, mortality rates were not high in this example and the lack of ahigh mortality rate in the challenge control group limits this parameteron which to base vaccine efficacy. Prior to Day 46, Groups 4 and 7 eachhad one out of fifteen pigs die, Group 9 had two out of fourteen pigsdie and Group 8 had three out of fifteen pigs die. Due to the fact thatGroup 9, the challenge control group was not demonstrating PCV2 clinicalsymptoms and only two deaths had occurred in this group by Day 46,Porcine Respiratory and Reproductive Syndrome Virus (PRRSV) MLV vaccinewas administered to all pigs on Day 46. Earlier studies had utilizedINGELVAC® PRRS MLV as an immunostimulant to exasperate PCV2-associatedPMWS disease and mortality rates were higher in these earlier studies.Two deaths occurred shortly after administering the PRRS vaccine on Day46—Group 4 had one death on Day 46 and Group 7 had one death on Day47—which were probably not associated with the administration of thePRRS vaccine. By Day 50, Group 8, which received two doses of killedvaccine, had the highest mortality rate (20%), followed by Group 9(challenge control) and Group 7 (0.25 ug rORF2—Carbopol), with mortalityrates of 14.3% and 13.3% respectively. Overall, administration of thePRRS vaccine to the challenge model late in the post-challengeobservation phase of this example did not significantly increasemortality rates.

Gross lesions in pigs with PMWS secondary to PCV2 infection typicallyconsist of generalized lymphadenopathy in combination with one or moreof the following: (1) interstitial pneumonia with interlobular edema,(2) cutaneous pallor or icterus, (3) mottled atrophic livers, (4)gastric ulcers, (5) nephritis and (6) reproductive disorders, e.g.abortion, stillbirths, mummies, etc. At necropsy (Day 50), icterus,hepatitis, and nephritis were not noted in any groups. A gastric ulcerwas noted in one Group 7 pig, but lymphadenopathy was not specificallyexamined for. Based on the presence of lesions that were consistent withPCV2 infection, three groups had at least one pig tentatively diagnosedwith PCV2 (PMWS). Group 8, which received two doses of killed vaccine,had 20% tentatively diagnosed with PCV2, while Group 7 and Group 4 had13.3% and 6.7%, respectively, tentatively diagnosed with PCV2. The mean% lung lesion scores varied between groups at necropsy. Groups 1, 2, 3,4, 6 and 10 had low % lung lesion scores that ranged from 0.11±0.38% to0.90±0.15%. As expected, Group 9, the challenge control group, had thehighest mean % lung lesion score (10.81±23.27%). In four groups, themean % lung lesion scores were elevated due to one to three pigs in eachof these groups having very high lung lesion scores. The lung lesionswere red/purple and consolidated. Typically, lung lesions associatedwith PMWS are described as tan, non-collapsible, and with interlobularedema. The lung lesions noted in this study were either not associatedwith PCV2 infection or a second pulmonary infectious agent may have beenpresent. Within the context of this study, the % lung lesion scoresprobably do no reflect a true measure of the amount of lung infectiondue to PCV2. Likewise, tentative diagnosis of pneumonia may have beenover-utilized as well. Any pigs with lung lesions, some as small as0.10% were listed with a tentative diagnosis of pneumonia. In thisexample, there was no sufficient difference between groups with respectto gross lesions and % lung lesions on which to base vaccine efficacy.

IHC results showed the largest differences between groups. Group 1 (16μg rORF2—IMS 1314) had the lowest positive IHC results for PCV2 antigen(0%); while Groups 9 and 10 had the highest positive IHC results withincidence rates of 100% and 93.3% respectively. Groups 3, 5, 6 and 7,which received 16, 4, 1 or 0.25 μg of rORF2 antigen, respectively,adjuvanted with Carbopol, had IHC positive rates of 20%, 20%, 40% and46.7%, respectively. Group 2, which received two doses of 16 μg vORF2adjuvanted with Carbopol had an IHC positive rate of 6.7%, while Group 4which received only one dose of the same vaccine, had an IHC positiverate of 13.3%. Due to the objective nature of this test and the factthat IHC results correlated with expected results, IHC testing isprobably one of the best parameters on which to base vaccine efficacy.

Thus in one aspect of the present invention, the Minimum ProtectiveDosage (MPD) of PCV2 rORF2 antigen adjuvanted with Carbopol in the CDCDpig model in the face of a PCV2 challenge is determined. Groups 3, 5, 6and 7 each received two doses of rORF2 antigen adjuvanted with Carbopol,but the level of rORF2 antigen varied for each group. Groups 3, 5, 6 and7 each received 16, 4, 1 or 0.25 μg of rORF2 antigen respectively. Ingeneral, decreasing the level of rORF2 antigen decreased PCV2 antibodytiters, and increased the mortality rate, mean % lung lesions, and theincidence of IHC positive tissues. Of the four groups receiving varyinglevels of rORF2—Carbopol, Groups 3 and 5, which received two doses of 16or 4 μg of rORF2 antigen, respectively, each had an IHC positive rate ofonly 20%, and each had similar antibody titers. Overall, based on IHCpositive results, the minimum protective dosage of rORF2 antigenadministered twice is approximately 4 μg.

In another aspect of the present invention, the antigenicity ofrecombinant (rORF2) and VIDO R-1 (vORF2) PCV2 antigens were assessed.Group 2 received two doses of 16 μg vORF2 and Group 3 received two dosesof 16 μg rORF2. Both vaccines were adjuvanted with Carbopol. Bothvaccines were found to be safe and both had 0% mortality rate. Group 2had a PCV2 antibody titer of 2507 on Day 25, while Group 3 had a PCV2antibody titer of 1503. Group 3 had a lower mean % lung lesion scorethan Group 2 (0.11±0.38% vs. 0.90±0.15%), but Group 2 had a lower IHCpositive incidence rate that Group 3 (6.7% vs. 20%). Overall, bothvaccines had similar antigenicity, but vORF2 was associated withslightly better IHC results.

In yet another aspect of the present invention, the suitability of twodifferent adjuvants (Carbopol and IMS 1314) was determined. Groups 1 and3 both received two doses of vaccine containing 16 ug of rORF2 antigen,but Group 1 received the antigen adjuvanted with IMS 1314 while Group 3received the antigen adjuvanted with Carbopol. Both groups hadessentially the same ADWG, essentially the same incidence of clinicalsigns post-challenge, the same mortality rate, and essentially the samemean % lung lesions; but Group 1 had an IHC positive rate of 0% whileGroup 3 had an IHC positive rate of 20%. However, Group 3, whichreceived the vaccine adjuvanted with Carbopol, had higher IFAT PCV2titers on Days 25, 32, and 50 than Group 1, which received the vaccineadjuvanted with IMS 1314. Overall, although the PCV2 vaccine adjuvantedwith IMS 1314 did provide better IHC results, it did not provideoverwhelmingly better protection from PCV2 infection and did induceinjection site reaction. Whereas the PCV2 vaccine adjuvanted withCarbopol performed nearly as well as the IMS 1314 adjuvanted vaccine,but was not associated with any adverse events.

In still another aspect of the present invention, the feasibility ofPCV2 ORF2 as a 1 ml, 1 dose product was determined. Groups 2 and 4 bothreceived 16 μg of vORF2 vaccine adjuvanted with Carbopol on Day 0, butGroup 2 received a second dose on Day 14. Group 4 had a slightly higherADWG and a lower mean % lung lesions than Group 2, but Group 2 hadhigher IFAT PCV2 titers on Day 25, 32 and 50, and a slightly lowerincidence rate of IHC positive tissues. All other results for these twogroups were similar. Overall, one dose of vORF2 adjuvanted with Carbopolperformed similar to two doses of the same vaccine.

1. A method for the prevention, lessening the severity of and/orreduction of Pia like lesions, normally known to be associated withLawsonia intracellularis infections in swine, comprising administeringto a swine at least one dose of an immunogenic composition of at least 2μg of recombinant PCV2 ORF2 protein.
 2. The method of claim 1, whereinsaid PCV2 ORF2 protein is i) a polypeptide comprising a sequenceselected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 9, SEQ ID NO: 10, and SEQ ID NO: 1; ii) any polypeptide that is atleast 80% homologous to the polypeptide of i), iii) any immunogenicportion of the polypeptides of i) and/or ii) iv) the immunogenic portionof iii), comprising at least 10, contiguous amino acids included in asequences selected from the group consisting of SEQ ID NO: 5, SEQ ID NO:6, SEQ ID No: 9, SEQ ID No: 10 and SEQ ID No: 11, v) a polypeptide thatis encoded by a DNA comprising the sequence of SEQ ID NO: 3 or SEQ IDNO:
 4. vi) any polypeptide that is encoded by a polynucleotide that isat least 80% homologous to the polynucleotide of v), vii) anyimmunogenic portion of the polypeptides encoded by the polynucleotide ofv) and/or vi) viii) the immunogenic portion of vii), whereinpolynucleotide coding for said immunogenic portion comprises at least 30contiguous nucleotides included in a sequence selected from the groupconsisting of SEQ ID No: 3, or SEQ ID No:
 4. 3. The method of claim 1,wherein said composition further comprises an additional componentselected from the group consisting of an inactivated viral vector, cellculture supernate, BEI, sodium thiosulfate, carriers, adjuvants, media,viral inactivators, diluents, isotonic agents, immunomodulatory agents,antibiotics, pharmaceutical acceptable salt, and combinations thereof.4. The method of claim 3, wherein said inactivated viral vector is arecombinant baculovirus coding for the PCV2 ORF2 protein.
 5. The methodof claim 3, wherein said adjuvant is selected from the group consistingof acrylic acid, methacrylic acid, and any polymer thereof.
 6. Themethod of claim 1, wherein said composition further comprises a polymerof an acrylic or methacrylic acid which is crosslinked with polyalkenylethers of sugars or polyalcohols.
 7. The method of claim 1, wherein saidcomposition further comprises a carbomer.
 8. The method of claim 7,wherein said carbomer is included in an amount between about 500 μg toabout 5 mg carbomer per dose.
 9. The method of claim 1, wherein one doseof said immunogenic composition of at least 4 μg of recombinant PCV2ORF2 protein is administered to the pig.
 10. The method of claim 1,wherein one dose of said immunogenic composition comprising at least 8μg of recombinant PCV2 ORF2 protein is administered to the pig.
 11. Themethod of claim 1, wherein one dose of said immunogenic compositioncomprising at least 16 μg of recombinant PCV2 ORF2 protein isadministered to the pig.
 12. The method of claim 1, wherein saidimmunogenic composition is effective for lessening the severity ofclinical symptoms associated with PCV2 infection comprising after asingle dose administration of said recombinant PCV2 ORF2 antigen. 13.The method of claim 1, wherein said immunogenic composition isadministered to pigs of 2 weeks of age or older.
 14. The method of claim1, wherein said immunogenic composition is administered to pigs notolder than 15 weeks of age.
 15. The method of claim 1, wherein one doseof the immunogenic composition has a volume of 1 ml.
 16. The method ofclaim 1, wherein said one dose of the immunogenic composition isadministered to said pig in 1 ml.
 17. The method of claim 1, whereinsaid one dose of the immunogenic composition is retained in a container.